Stilbenoid prenyltransferases from plants

ABSTRACT

The process and system led to the identification of prenyltransferase genes from elicitor-treated peanut hairy roots. One of the prenyltransferases, AhR4DT-1 catalyzes a key reaction involved in the biosynthesis of prenylated stilbenoids, in which resveratrol is prenylated at its C-4 position to form arachidin-2, while another, AhR3′DT-1, was able to add the prenyl group to C-3′ of resveratrol. Each of these prenyltransferases has a high specificity for stilbenoid substrates, and their subcellular location in the plastid was confirmed by fluorescence microscopy. Structure analysis of the prenylated stilbenoids suggest that these two prenyltransferase activities represent the first committed steps in the biosynthesis of a large number of prenylated stilbenoids and their derivatives in peanut.

CROSS-REFERENCE TO RELATED APPLICATIONS

This application claims priority to and is a continuation in part ofU.S. Patent Application No. 62/351,117 filed on Jun. 16, 2016 entitled“STILBENOID PRENYLTRANSFERASES FROM PEANUT.”

STATEMENT REGARDING FEDERALLY SPONSORED RESEARCH OR DEVELOPMENT

This work was supported by USDA-NIFA Grant 2014-67014-21701, NationalSciences Foundation-EPSCoR Grant EPS 0701890 (Center for Plant-PoweredProduction-P3), the National Science Foundation Division of BiologicalInfrastructure Grant ABI-1062432 to Indiana University (National Centerfor Genome Analysis Support), the Arkansas ASSET Initiative, theArkansas Science & Technology Authority, and the Arkansas BiosciencesInstitute.

REFERENCE TO SEQUENCE LISTING SUBMITTED VIA EFS-WEB

The official copy of the sequence listing is submitted electronicallyvia EFS-WEB as an ASCII (.txt) formatted sequence listing with a filenamed 8-26-19-SEQ ST25.txt, created on Aug. 28, 2019 and having a sizeof 72 KB and accompanies this specification. The sequence listingcontained in this ASCII formatted document is part of the specificationand is herein incorporated by reference in its entirety and includes nonew matter.

REFERENCE TO A MICROFICHE APPENDIX

Not Applicable.

RESERVATION OF RIGHTS

A portion of the disclosure of this patent document contains materialwhich is subject to intellectual property rights such as but not limitedto copyright, trademark, and/or trade dress protection. The owner has noobjection to the facsimile reproduction by anyone of the patent documentor the patent disclosure as it appears in the Patent and TrademarkOffice patent files or records but otherwise reserves all rightswhatsoever.

BACKGROUND OF THE INVENTION

The present invention relates generally to prenylated stilbenoids andthe production of prenylated stilbenoids.

Description of the Known Art

A substantial part of non-host defense responses in many plants is thepathogen-induced production of secondary metabolites, generally termedphytoalexins, that locally restrict disease progression due tobioactivities toxic to the pathogen (reviewed in Ahuj a et al., 2012).Peanut or groundnut (Arachis hypogaea) tissues mount a defense againstinfection by the soil fungus Aspergillus flavus and other pathogens byoverproducing stilbene derivatives around sites of wounding and elicitorperception (Sobolev, 2013).

SUMMARY OF THE INVENTION

The present invention is related to prenylated stilbenoids and a methodof producing the prenylated stilbenoids. Harnessing the induciblebioproduction capabilities of the peanut hairy root culture system, wehave newly produced a prenylated stilbenoid, i.e., arachidin-5, and havedemonstrated that the prenyl moiety on peanut prenylated stilbenoids isderived from a plastidic biosynthesis pathway. We have characterized forthe first time plant membrane-bound stilbenoid-specificprenyltransferase activity from the microsomal fraction of peanut hairyroots. With multidisciplinary approaches, we have isolated stilbenoidprenyltransferase genes and comprehensively characterized theirfunctionality as stilbenoid prenyltransferase via a transient expressionsystem in Nicotiana benthamiana and stable expression in tobacco plantsand hairy roots. Moreover, we have observed the enzymatic degradation ofexogenous resveratrol by peanut hairy root tissue, an observation thatwill lead to elucidation of further mechanisms governing phytoalexinaccumulation in plants.

Prenylated stilbenoids synthesized in some legumes exhibit plantpathogen defense properties and pharmacological activities withpotential benefits to human health. Despite their importance, thebiosynthetic pathways of these compounds remain to be elucidated. Peanut(Arachis hypogaea) hairy root cultures produce a diverse array ofprenylated stilbenoids upon treatment with elicitors. Using metabolicinhibitors of the plastidic and cytosolic isoprenoid biosyntheticpathways, we demonstrated that the prenyl moiety on the prenylatedstilbenoids derives from a plastidic pathway. We further characterizedfor the first time a membrane-bound stilbenoid-specificprenyltransferase from the microsomal fraction of peanut hairy roots.This microsomal fraction-derived prenyltransferase utilizes3,3-dimethylallyl pyrophosphate as a prenyl donor. The microsomalfraction also prenylates pinosylvin to chiricanine A and piceatannol toarachidin-5, a prenylated stilbenoid produced for the first time in thisstudy. Using a transcriptomics and targeted metabolomics approach, wehave isolated stilbenoid prenyltransferase genes from peanut andconfirmed their functionality as stilbenoid prenyltransferase via atransient expression system in Nicotiana benthamiana and stableexpression in tobacco plants and hairy roots.

Defense responses of peanut (Arachis hypogaea) include the synthesis ofprenylated stilbenoids as phytoalexins in response to biotic and abioticstresses. Despite their importance, the biosynthetic pathways ofprenylated stilbenoids remain to be elucidated and genes encodingstilbenoid-specific prenyltransferases have yet to be identified in anyplant species. In this study, we combined targeted transcriptome andmetabolome analyses to discover prenyltransferase genes fromelicitor-treated peanut hairy roots. Transcripts encoding five enzymeswere identified, and two of these were functionally characterized in atransient expression system of Agrobacterium-infiltrated leaves ofNicotiana benthamiana. One of the prenyltransferases, AhR4DT-1 catalyzesa key reaction involved in the biosynthesis of prenylated stilbenoids,in which resveratrol is prenylated at its C-4 position to formarachidin-2, while another, AhR3′DT-1, was able to add the prenyl groupto C-3′ of resveratrol. Each of these prenyltransferases has a highspecificity for stilbenoid substrates, and their subcellular location inthe plastid was confirmed by fluorescence microscopy. Structure analysisof the prenylated stilbenoids suggest that these two prenyltransferaseactivities represent the first committed steps in the biosynthesis of alarge number of prenylated stilbenoids and their derivatives in peanut.

Stilbenoids are phenylpropanoid compounds that accumulate in response tobiotic and abiotic stresses in a small number of higher plant familiesincluding grape (Vitaceae), pine (Pinaceae) and peanut (Fabaceae). Thesecompounds serve as phytoalexins and provide protection to the host plantagainst various microbial pathogens (Ahuja et al., 2012). Resveratrol(3,5,4′-trihydroxy-trans-stilbene), as the most studied compound in thestilbene family has attracted great attention in the scientificcommunity, not only because of its important role for disease defense inplants (Ahuja et al., 2012), but also because of numerous bioactivitiesincluding anticancer, cardioprotective, antioxidant, anti-inflammatoryand neuroprotective properties in human cell culture and in vivo,although the extent of its bioavailability has been questioned (Gambiniet al., 2015; Tome-Carneiro et al., 2013; Baur and Sinclair, 2006).

Interestingly, resveratrol is synthesized in peanut, along withstilbenoids conjugated to a prenyl group, a modification not common inother stilbene-producing plants (Aguamah et al., 1981; Cooksey et al.,1988; Sobolev et al., 2006). The prenylation of the stilbene backbone isthe primary feature that contributes to the diversity of these peanutsecondary metabolites. Differences occur in the position of prenylationas well as in subsequent modifications of the prenyl moiety, such ascyclization and hydroxylation. To date, more than 45 prenylatedstilbenoids and their derivatives have been detected in peanut tissuessubjected to biotic or abiotic stress (Wu et al., 2011; Sobolev, 2013;Sobolev et al., 2010, 2009, 2006, 2016). The prenylation of stilbenoidsincreases their lipophilicities and membrane permeabilities, and mayhave additional impacts on bioactivities. Prenylated stilbenoids haveshown equivalent or enhanced bioactivities relative to non-prenylatedforms, such as resveratrol, in in vitro studies (Huang et al., 2010;Chang et al., 2006; Sobolev et al., 2011). The prenylated stilbenoidsarachidin-1 and arachidin-3 showed favorable metabolic profiles whencompared to their non-prenylated analogs piceatannol and resveratrol(Brents et al., 2012). Furthermore, arachidin-1 and arachidin-3exhibited specific bioactivities not found in their non-prenylatedforms, such as inhibiting the replication of rotavirus in HT29.f8 cells(Ball et al., 2015). Also, prenylated stilbenoids showed higher affinityto human cannabinoid receptors when compared to non-prenylatedstilbenoids (Brents et al., 2012).

Prenyltransferase(s) responsible for these stilbenoid modificationsis/are crucial for the biosynthesis of peanut bioactive compounds ofinterest. To date however, no gene encoding a stilbenoidprenyltransferase has been identified in plants. Previously, wedeveloped a peanut hairy root culture system to serve as a platform forsustainable production of prenylated stilbenoids (Yang et al., 2015;Condori et al., 2010). Leveraging this system more recently, wecharacterized biochemically the first stilbenoid-specificprenyltransferase activity from the microsomal fraction of theseelicited cultures (Yang et al., 2016). This prenyltransferase utilizesplastid-derived dimethylallyl pyrophosphate (DMAPP) to prenylateresveratrol or piceatannol into arachidin-2 or arachidin-5,respectively, and shares several features in common with flavonoidprenyltransferases reported in other legume species. For instance, allidentified flavonoid and stilbenoid prenyltransferase enzymaticreactions require divalent cations as cofactors, and show maximumactivity at basic pH (Sasaki et al., 2008; Akashi et al., 2008; Sasakiet al., 2011; Shen et al., 2012; Li et al., 2014; Chen et al., 2013;Yang et al., 2016). These characteristics and others guided cloning ofthe peanut prenyltransferase genes described here. We took a dualapproach, combining parallel targeted transcriptome and metabolomeanalyses to isolate prenyltransferase activities. We generated extensiveRNAseq data assemblies designed to capture elicitor-induced mRNAs, andused these sequences to clone potential prenyltransferase cDNAs. Here wedescribe the first transcripts to be identified as encodingstilbenoid-specific prenyltransferase enzymes. These catalyze twodistinct dimethylallylation reactions in the biosynthesis of prenylatedstilbenoids. Functionalities of the enzymes are uncovered using atransient expression system of Agrobacterium-infiltrated Nicotianabenthamiana leaves, and their subcellular location is proposed fromfluorescence microscopy imaging in particle-bombarded onion epidermalcells. We further use available genome assemblies of the two diploidprogenitors of A. hypogaea to align the prenyltransferase transcriptsand estimate their genomic structure.

It is an object of the present invention to isolate or purify nucleicacid molecules comprising a gene sequence that encodes a polypeptidehaving stilbenoid prenyltranferase activity.

It is another object of the present invention to produce a prenylatedstilbenoid in an organism, cell or tissue, said method comprising theexpression or over-expression of a gene that encodes for a stilbenoidprenyltransferase.

It is another object of the present invention to produce a prenylatedstilbenoid in an organism wherein the organism is peanut, tobacco,grape, muscadine, Polygonum, pine, or yeast.

It is another object of the present invention to produce a prenylatedstilbenoid in an organism wherein the method comprises expressing a genesequence that encodes a polypeptide having stilbenoid prenyltransferaseactivity in an organism, including but not limited to, peanut, tobacco,grape, muscadine, Polygonum, pine, or yeast.

It is another object of the present invention to provide a newprenylated stilbenoid.

It is another object of the present invention to provide an improvedmethod of producing a prenylated stilbenoid.

It is another object of the present invention to provide a method ofproducing the improved stilbenoid.

It is another object of the present invention to produce the stilbenoidin greater quantities.

It is another object of the present invention to provide a moreeffective stilbenoid.

It is another object of the present invention to provide a method ofproducing the more effective stilbenoid.

These and other objects and advantages of the present invention, alongwith features of novelty appurtenant thereto, will appear or becomeapparent by reviewing the following detailed description of theinvention.

BRIEF DESCRIPTION OF THE DRAWINGS

In the following drawings, which form a part of the specification andwhich are to be construed in conjunction therewith, and in which likereference numerals have been employed throughout wherever possible toindicate like parts in the various views:

FIG. 1 shows A, Structure of stilbene backbone and main prenylationpatterns found on peanut prenylated stilbenoids. B, Chemical structuresof stilbenoids identified in elicited peanut hairy roots: (a)resveratrol; (b) piceatannol; (c) arachidin-2; (d) arachidin-5; (e)arachidin-3; (f) arachidin-1. All compounds are shown in their transisomers.

FIG. 2 shows a HPLC chromatogram (UV 320 nm) of ethyl acetate extractfrom peanut hairy root culture medium after 72 h post treatment with 100μM methyl jasmonate and 9 g/L methyl-β-cyclodextrin. a, arachidin-5derivative; b, arachidin-2 derivative.

FIG. 3 shows effects of mevastatin and clomazone on the production ofstilbenoids in elicited peanut hairy root culture. A, HPLC chromatograms(UV 340 nm) of ethyl acetate extracts from peanut hairy root culturesafter 48-hour treatment with (a) 100 μM methyl jasmonate (MeJA) and 9g/L methyl-β-cyclodextrin (CD); (b) 100 μM MeJA, 9 g/L CD, and 10 μMmevastatin; (c) 100 μM MeJA, 9 g/L CD, and 10 μM clomazone and (d) 100μM MeJA, 9 g/L CD, and 100 μM clomazone. B, Yields of resveratrol,arachidin-2, arachidin-3, piceatannol, arachidin-5, and arachidin-1after 48 hour and 72 hour co-treatment of 10 μM mevastatin, 10 μM or 100μM clomazone with elicitors (CD+MeJA). Values shown are the average mMyield of three replicates and error bars represent standard deviation.The asterisks above bars represent significant difference when comparedto the group treated with MeJA and CD alone (*, p<0.05; **, p<0.01; ***,p<0.001; ****, p<0.0001; n.s., not significant; n.d., not detected); andthe asterisks above the connecting line represent significant differencebetween 48 hour and 72 hour treatments (*, p<0.05; ***, p<0.001; ****,p<0.0001; n.s., not significant). Statistical analyses were performed bytwo-way ANOVA with Dunnett's and Sidak's multiple-comparisons testrespectively.

FIG. 4 shows enzymatic degradation of resveratrol by crude cell-freeprotein extract from non-elicited peanut hairy root. HPLC chromatograms(UV 320 nm) of ethyl acetate extracts from the incubation mixturescontained 1 mM resveratrol with (A) 50 μg crude cell-free proteinextract for 30 min; (B) 50 μg heat-denatured crude cell-free proteinextract for 30 min; (C) 50 μg crude cell-free protein extract andadditional 5 mM DTT for 120 min. All reactions were done in a pH 7.6Tris-HCl buffer. (D) Close-up (3.92-7.19 min) of chromatogram shown in(A).

FIG. 5 shows resveratrol prenyltransferase activity in microsomalfraction of elicited peanut hairy root. HPLC chromatograms (UV 320 nm)of (A) purified arachidin-2; (B) ethyl acetate extract of 500 μlreaction mixture containing 30 μg microsomal fraction, 100 μMresveratrol, 300 μM DMAPP, 10 mM MgCl₂ and 5 mM DTT (C) ethyl acetateextract of 500 μl reaction mixture containing heat-denatured 30 μgmicrosomal fraction, 100 μM resveratrol, 300 μM DMAPP, 10 mM MgCl₂ and 5mM DTT. All reactions were done for 60 min in a pH 9.2 Tris-HCl buffer.

FIG. 6 shows substrate specificity of resveratrol prenyltransferase inmicrosomal fraction of elicited peanut hairy root. A, Chemicalstructures of prenyl acceptors used for substrate specificity analysisand their prenylated products: stilbenoids (a1, resveratrol, a2arachidin-2; b1, piceatannol, b2, arachidin-5; c1, pinosylvin, c2,chiricanine-A, d, oxyresveratrol and e, pterostilbene), flavanone (e,naringenin), flavone (f, apigenin) and isoflavone (g, genistein). B,Prenylation activity of microsomal fraction with various prenylacceptors. Values are the average of 2 replicates and error barsrepresent standard deviation (n.d., not detected).

FIG. 7 shows a table of detected stilbenoids.

FIG. 8 shows a table of resveratrol prenyltransferase activity.

FIG. 9 shows enzymatic characterization of AhR4DT transiently expressedin Nicotiana benthamiana leaf HPLC chromatograms (UV 320 nm) of ethylacetate extraction of 1000 μl reaction mixture of 200 μM resveratrol,600 μM DMAPP, 10 mM MgCl₂ and 10 mM DTT incubated with 50 μl crudeprotein of Nicotiana benthamiana leaf after vacuum infiltration withAgrobacterium tumefaciens LBA4404 harboring pBIBKan-9b13 binary vectorfor (A) 48 hours and (B) 72 hours in a pH 9.2 Tris-HCl buffer for 40min. Chromatograms of the reactions with (C) heated denatured protein)and (D) no DMAPP added for control.

FIG. 10 shows enzymatic characterization of resveratrolprenyltransferase transiently expressed in Nicotiana benthamiana leafHPLC chromatograms (UV 320 nm) of ethyl acetate extraction of 1000 μlreaction mixture of 200 μM resveratrol, 600 μM DMAPP, 10 mM MgCl₂ and 10mM DTT incubated with 50 μl crude protein of Nicotiana benthamiana leafafter vacuum infiltration with Agrobacterium tumefaciens LBA4404harboring (A) pBMKan-10k1; (B) pBIBKan-4e1; (C) pBIBKan-4e10 and (D)pBIBKan-5m3 binary vectors in a pH 9.2 Tris-HCl buffer for 40 min.

FIG. 11 shows the development of transgenic tobacco plants expressing astilbenoid-specific prenyltransferase from peanut. A, B: Regeneratedshoots from the Agrobacterium inoculation sites. C: Rooting oftransgenic tobacco plant.

FIG. 12 shows a sequence view of the nucleotide sequence (SEQ ID NO:3)and amino acid sequence (SEQ ID NO:2) of stilbenoid-specificprenyltransferase AhR4DT-9b13 (renamed as AhR4DT-1; GenBank accessionNo. KY565244) (SEQ ID NO:1) involved in the biosynthesis of arachidin-2.

FIG. 13 shows a sequence view of the nucleotide sequence (SEQ ID NO:6)and amino acid sequence (SEQ ID NO:5) of stilbenoid prenyltransferaseAhRPT-4e1 (Renamed as AhR3′DT-2; GenBank Accession No. KY565246) (SEQ IDNO:4).

FIG. 14 shows a sequence view of the nucleotide sequence (SEQ ID NO:9)and amino acid sequence (SEQ ID NO:8) of stilbenoid prenyltransferaseAhRPT-4e10 (Renamed as AhR3′DT-3; GenBank Accession No. KY565247).

FIG. 15 shows a sequence view of the nucleotide sequence (SEQ ID NO:12)and amino acid sequence (SEQ ID NO:11) of stilbenoid prenyltransferaseAhRPT-5m3 (Renamed as AhR3′DT-4; GenBank Accesion No. KY565248) (SEQ IDNO:10).

FIG. 16 shows a sequence view of the nucleotide sequence (SEQ ID NO:15)and amino acid sequence (SEQ ID NO:14) of stilbenoid prenyltransferaseAhRPT-10k1 (AC1) (Renamed as AhR3′DT-1; GenBank Accesion No. KY565245)(SEQ ID NO:13).

FIG. 17 shows ¹H NMR analysis of arachidin-5. ¹H NMR was recorded at 400MHz in acetone-d₆ on a Bruker AV-400 NMR spectrometer.

FIG. 18 shows ¹³C NMR analysis of arachidin-5. ¹³C NMR was recorded at100 MHz in acetone-d₆ on a Bruker AV-400 NMR spectrometer.

FIG. 19 shows effect of mevastatin on the production of stilbenoids inelicited peanut hairy root culture. HPLC chromatograms (UV 340 nm) ofethyl acetate extracts from peanut hairy root cultures after 48-hourtreatment with (A) 100 μM methyl jasmonate (MeJA) and 9 g/Lmethyl-β-cyclodextrin (CD); (B) 100 μM MeJA, 9 g/L CD, and 1 μMmevastatin; (C) 100 μM MeJA, 9 g/L CD, and 10 μM mevastatin and (D) 100μM MeJA, 9 g/L CD, and 100 μM mevastatin.

FIG. 20 shows effect of clomazone on the production of stilbenoids inelicited peanut hairy root culture. HPLC chromatograms (UV 340 nm) ofethyl acetate extracts from peanut hairy root cultures after 48-hourtreatment with (A) 100 μM methyl jasmonate (MeJA) and 9 g/Lmethyl-β-cyclodextrin (CD); (B) 100 μM MeJA, 9 g/L CD, and 1 μMclomazone; (C) 100 μM MeJA, 9 g/L CD, and 10 μM clomazone and (D) 100 μMMeJA, 9 g/L CD, and 100 μM clomazone.

FIG. 21 shows effect of clomazone on the production of stilbenoids innon-elicited peanut hairy root culture. HPLC chromatograms (UV 340 nm)of ethyl acetate extracts from peanut hairy root cultures after 48-hourtreatment with (A) 100 μM methyl jasmonate and 9 g/Lmethyl-β-cyclodextrin; (B) 10 μM clomazone; (C) 50 μM clomazone and (D)100 μM clomazone.

FIG. 22 shows comparison of stilbenoid yields from the culture mediumand root tissue. Ethyl acetate extracts were prepared from elicitedpeanut hairy root cultures. HPLC chromatograms (UV 340 nm) of ethylacetate extract from (A) culture medium and (B) lyophilized root tissue.Cultures were elicited for 48 hours with 100 μM methyl jasmonate and 9g/L methyl-β-cyclodextrin.

FIG. 23 shows time course of resveratrol prenyltansferase assay withoutDTT and subsequent degradation of resveratrol and arachidin-2 in thereaction. Assays were done for 0 to 120 min. HPLC chromatograms (UV 320nm) of ethyl acetate extracts from the reaction mixture of 30 μgmicrosomal fraction, 100 μM resveratrol, 300 μM DMAPP, and 10 mM MgCl₂.Assays were done for 0, 10, 20, 30, 45, 60 or 120 min in a pH 9.2Tris-HCl buffer.

FIG. 24 shows biotransformation of arachidin-2 by protein extracts fromelicited peanut hairy root culture. HPLC chromatograms (UV 320 nm) ofethyl acetate extracts from the 60 min reaction contained (a) 100 μMarachidin-2 with 30 μg heat-denatured crude cell-free protein extract ascontrol; (b) 30 μg crude cell-free protein extract; (c) 30 μg microsomalfraction and (d) 30 μg 156,000 g supernatant. All reactions were done ina pH 9.2 Tris-HCl buffer.

FIG. 25 shows resveratrol prenyltransferase activity. A, Concentrationsof remaining resveratrol and generated arachidin-2 from the reactionmixtures with varying incubation times (30, 60, 90 and 120 min) weremeasured by HPLC. B, Concentrations of remaining resveratrol andgenerated arachidin-2 from the reaction mixtures with varying amounts ofmicrosomal fraction (30, 60, 90 and 120 μg) were measured by HPLC.

FIG. 26 shows pH dependency of resveratrol prenyltransferase activity.Resveratrol prenyltransferase activity at various pH values was measuredusing 100 mM Tris-HCl buffer at pH 7.0, 8.0, 8.4, 8.6, 8.8, 9.0, 9.2,9.4, 9.6 and 10.

FIG. 27 shows divalent cation requirement for resveratrolprenyltransferase activity.

Resveratrol prenyltransferase activity was compared in the presence ofvarious divalent cations: Mg²⁺, Mn²⁺, Fe²⁺, Ca²⁺, Co²⁺, zn²⁺, Ni²⁺ andCu²⁺. The activity in the presence of Mg²⁺ is shown as 100%.

FIG. 28 shows biochemical characterization of resveratrolprenyltransferase in microsomal fraction of elicited peanut hairy root.HPLC chromatogram (UV 320 nm) of ethyl acetate extract of a 60 minincubation mixture containing (A) standard reaction (30 μg microsomalfraction, 100 μM resveratrol, 300 μM DMAPP, 10 mM MgCl₂ and 5 mM DTT ina pH 9.2 Tris-HCl buffer); (B) standard reaction without divalent cationadded; (C) standard reaction with 300 μM IPP instead of DMAPP and (D)standard reaction with 30 μg microsomal fraction isolated fromnon-elicited peanut hairy root instead of 30 μs microsomal fractionisolated from elicited peanut hairy root.

FIG. 29 shows effects of (A) resveratrol and (B) DMAPP concentrations onresveratrol prenyltransferase activity. Enzymatic activity was measuredwith a microsomal fraction from peanut hairy roots. The apparent K_(m)and V_(max) values for resveratrol and DMAPP were determined withvarying concentrations of resveratrol (10-640 μM) and of DMAPP (10-640μM) respectively and calculated via nonlinear regression analysis withMichaelis-Menten equation by Graphpad Prism 6 software.

FIG. 30 shows substrate specificity of resveratrol prenyltransferase inmicrosomal fraction of elicited peanut hairy root. HPLC chromatograms(UV 320 nm) of ethyl acetate extracts of a 60 min incubation mixturecontaining 30 μg microsomal fraction with 300 μM DMAPP, 10 mM MgCl₂ and5 mM DTT, and 100 μM prenyl acceptors (A, resveratrol; B, piceatannol;C, pinosylvin; D, pterostilbene; E, naringenin; F, apigenin and G,genistein) in a pH 9.2 Tris-HCl buffer.

FIG. 31 shows biotransformation of arachidin-5 by protein fractions fromelicited peanut hairy root culture. HPLC chromatograms (UV 320 nm) ofethyl acetate extracts from the 60 min incubation mixtures contained 40μM arachidin-5 with (A) 30 μg heated microsomal fraction as control and(B) 30 μg microsomal fraction. All reactions were done in a pH 9.2Tris-HCl buffer.

FIG. 32 shows substrate specificity of resveratrol prenyltransferase inmicrosomal fraction of elicited peanut hairy root. A, Chemicalstructures of oxyresveratrol and its prenylated product. B, HPLCchromatograms (UV 320 nm) of ethyl acetate extraction of reactionmixtures contained 100 μM oxyresveratrol, 300 μM DMAPP, 10 mM MgCl₂, 5mM DTT and 30 microsomal fraction (above); heated denatured microsomalfraction (below) in a pH 9.2 Tris-HCl buffer for 60 min. C,HPLC-PDA-ESI-MS³ analysis of oxyresveratrol, prenylated oxyresveratroland its derivative.

FIG. 33 shows enzymatic characterization of stilbenoid prenyltransferasegenes (AhR4DT-9b13, AhRPT-10k1, AhRPT-4e1, AhRPT-4e10, and AhRPT5m3)transiently expressed in Nicotiana benthamiana leaf HPLC chromatograms(UV 320 nm) of ethyl acetate extraction of 1000 μl reaction mixture of200 μM resveratrol, 600 μM DMAPP, 10 mM MgCl₂ and 10 mM DTT incubatedwith 50 μl crude protein of Nicotiana benthamiana leaf after vacuuminfiltration with Agrobacterium tumefaciens LBA4404 harboringpBMKan-9b13 (A); pB1I3Kan-10k1 (B); pBIBKan-4e1 (C); pBIBKan-4e10 (D);pBIBKan-5m3 (E) and pBIBKan (F) binary vectors in a pH 9.2 Tris-HClbuffer for 40 min.

FIG. 34 shows resveratrol prenyltransferase activities in transgenictobacco (Nicotiana tabacum) expressing AhRPT-10k1 gene cloned frompeanut hairy roots. HPLC chromatograms (UV 320 nm) of ethyl acetateextraction of 500 μl reaction mixture of 200 μM resveratrol, 300 μMDMAPP, 10 mM MgCl₂ and 10 mM DTT incubated with 50 μg crude protein oftransgenic N. tabacum leaf expressed 10k1 gene (A) and 50 μg ofmicrosomal fraction of transgenic N. tabacum hairy roots expressed 10k1gene (B) for 2 hours.

FIG. 35 shows the enzymatic characterization of recombinant stilbenoidprenyltransferases expressed in Nicotiana benthamiana. (A) AhR4DT-1 andAhR′3DT-1/2/3/4 from peanut catalyzes the 4 and 3′-prenylation ofresveratrol, respectively. (B) Enzymatic characterization of resveratrolprenyltransferase transiently expressed in Nicotiana benthamiana leaf.HPLC chromatograms (UV 320 nm) of ethyl acetate extract of 1 mL reactionmixture of 100 μM resveratrol, 300 μM DMAPP, 10 mM MgCl₂ and 10 mM DTTincubated with 5 mg crude protein of N. benthamiana leaf after vacuuminfiltration with Agrobacterium tumefaciens LBA4404 harboring (B)pBIB-Kan-AhR4DT-1; (C) pBIB-Kan-AhR3′DT-1; (D) pBIB-Kan-AhR3′DT-2; (E)pBIB-Kan-AhR3′DT-3; (F) pBIB-Kan-AhR3′DT-4 and (G) pBIB-Kan binaryvectors in a pH 9.0 Tris-HCl buffer for 40 min.

FIG. 36 shows the primary structures of stilbenoid prenyltransferases.Two conserved NQXXDXXXD (SEQ ID NO:16) and KDXXDXEGD (SEQ ID NO:17)motifs are boxed.

FIG. 37 shows the phylogenetic relationship between peanut stilbenoidprenyltransferases and related prenyltransferases accepting aromaticsubstrates. Species abbreviations are: Aa, Allium ampeloprasum; Ah,Arachis hypogaea; At, Arabidopsis thaliana; Cl, Citrus limon; Cp, Cupheaavigera var pulcherrima; Cr, Chlamydomonas reinhardtii; Ct, Cudraniatricuspidata; Gm, Glycine max; H1, Humulus lupulus; Hv, Hordeum vulgare;La, Lupinus albus; Le, Lithospermum erythrorhizon; Ma, Morus alba; Os,Oryza sativa; Pc, Petroselinum crispum; Sf, Sophora flavescens; Ta,Triticum aestivum; Zm, Zea mays. Homogentisate phytyltransferases(VTE2-1s) and homogentisate geranylgeranyltransferases (HGGTs),homogentisate solanesyltransferases (VTE2-2s), p-hydroxybenzoategeranyltransferase (PGT) andp-hydroxybenzoate polyprenyltransferases(PPTs) are involved in the biosynthesis of vitamin E, plastoquinone,shikonin and ubiquinone, respectively. IDT is isoliquiritigenin dimethylallyltransferase. Accession numbers of these proteins are listed in FIG.69.

FIG. 38 shows the substrate specificity of AhR4DT-1 and AhR3′DT-1. A,Chemical structures of prenyl acceptors used for substrate specificityanalysis and their prenylated products: stilbenoids (a, resveratrol, b,piceatannol, c, oxyresveratrol, d, pinosylvin, e, piceid and f,pterostilbene), flavanone (g, naringenin), flavone (h, apigenin) andisoflavone (i, genistein). B, Relative prenylation activity of AhR4DT-1(B) and AhR3′DT-1 (C) with various prenyl acceptors were compared withthat of resveratrol. The prenyl donor specificity of AhR4DT-1 (D) andAhR3′DT-1 (E) were tested using DMAPP, IPP, GPP, FPP, GGPP withresveratrol as a prenyl acceptor. All these reactions were performed in100 mM Tric-HCl buffer (pH 9.0) at 28° C. for 40 mins. Values are theaverage of triplicate and error bars represent standard deviation (n.d.,not detected).

FIG. 39 shows the microscopic analysis of subcellular localization ofAhR4DT-1 and AhR3′DT-1 fused with GFP in onion epidermal cells uponparticle bombardment. (A, D) Plasmids containing CaMV735S-TEV-GFP andRS-TP-mCherry, (B, E) AhR4DT-1-GFP and RS-TP-mCherry or (C, F)AhR3′DT-1-GFP and RS-TP-mCherry were mixed and introduced into onionepidermal cells by particle bombardment.

Magnification: 20×; Scale bars in A, B and C equal 100 μm.

A1, B1, C1: Bright field images of onion cells.

A2, B2, C2: Expression pattern of mCherry from constructCaMV35S-RS-TP-mCherry

A3: Expression pattern of GFP from construct CaMV35S-TEV-GFP

B3: Expression pattern of GFP from construct AhR4DT-1-GFP

C3: Expression pattern of GFP from construct AhR3′DT-1-GFP

A4: Merge of A2 and A3

B4: Merge of B2 and B3

C4: Merge of C2 and C3

Close up of the same cells shown. Scale bars in D, E and F equal 10 μm

D1, E1, F1: Expression pattern of mCherry from constructCaMV35S-RS-TP-mCherry

D2: Expression pattern of GFP from construct CaMV35S-TEV-GFP

E2: Expression pattern of GFP from construct AhR4DT-1-GFP

F2: Expression pattern of GFP from construct AhR3′DT-1-GFP

D3: Merge of D1 and D2

E3: Merge of E1 and E2

F3: Merge of F1 and F2

CaMV35S: cauliflower mosaic virus 35S promoter; TEV: tobacco etch virustranslational enhancer; GFP: green fluorescent protein; RS-TP: rubiscosmall subunit transit peptide.

FIG. 40 shows the enzyme activities and transcript coexpression duringelicitation time course in peanut hairy root cultures. (A)Prenyltransferase activities from crude cell-free extracts. (B) Relativetranscript accumulation of AhR4DT-1 and (C) AhR3′DT-1 as determined byRT-qPCR. (D) Uniquely mapped RNA-Seq reads coverage over reference A.hypogaea transcripts AhR4DT-1, (E) AhR3′DT-1, (F) AhR3′DT-2/3 and (G)AhR3′DT-4 as described. E9 and E72, 9 h and 72 h MeJA+CD treatment; C9and C72, 9 h and 72 h control treatments.

FIG. 41 shows the proposed pathway of prenylated stilbenoids in peanut.Stilbenoids identified from the medium of peanut hairy root culture uponelicitors treatment are bolded and their proposed pathway is highlightedin yellow. Other prenylated stilbenoids identified in fungal-challengedpeanut seeds are divided into three groups based on the prenyl unit andhydroxyl groups on their stilbene backbone. Prenylation reactionscatalyzed by AhR4DT-1 and AhR3′DT-1 identified in this study are labeledwith red solid arrows. Enzymatic reactions confirmed in peanut aremarked with black solid arrow, while other proposed reactions arelabeled in black arrows with dashed lines. *: Pinosylvin,3-methyl-2-butenyl-3′-resveratrol and the prenylation product ofpiceatannol by AhR3′DT-1 have not been reported in peanut tissue.

FIG. 42 shows the prenylation of resveratrol by AhR3′DT-1.Prenyltransferase activity of AhR3′DT-1 in microsomal fraction ofNicotiana benthamiana leaf after vacuum infiltration with Agrobacteriumtumefaciens LBA4404 harboring pBIB-Kan-AhR3′DT-1 was confirmed by HPLCand LC-MS^(n) analysis.

(A) Chemical structures of resveratrol and its prenylated product,3-methyl-2-butenyl-3′-resveratrol.

(B) HPLC chromatograms (UV 320 nm) of ethyl acetate extraction ofreaction mixtures contained 100 μM resveratrol, 300 μM DMAPP, 10 mMMgCl₂, 5 mM DTT and 30 microsomal fraction in a pH 9.0 Tris-HCl bufferfor 40 min.

(C) HPLC-PAD-ESI-MS³ analysis of prenylated product,3-methyl-2-butenyl-3′-resveratrol.

FIG. 43 shows NMR spectra of the 1 mM 3-methyl-2-butenyl-3′-resveratrolisolated from a large-scale enzymatic assay.

1D ¹H NMR (A), 1D ¹³C NMR spectrum (B), 2D ¹H-¹³C HMBC (C) and 2D ¹H-¹³CHSQC (D) spectra obtained on 700 MHz Bruker Avance spectrometerdissolved in d6-Acetone at 298K. The peaks circled in red are consistentin both 1D and 2D experiments which represent protons on the specificlocations of the scaffold.

FIG. 44 shows Prenylation of resveratrol by AhR4DT-1.

Prenyltransferase activity of AhR4DT-1 in microsomal fraction ofNicotiana benthamiana leaf after vacuum infiltration with Agrobacteriumtumefaciens LBA4404 harboring pBIB-Kan-AhR4DT-1 was confirmed by HPLCand LC-MS^(n) analysis.

(A) Chemical structures of resveratrol and its prenylated product,arachidin-2.

(B) HPLC chromatograms (UV 320 nm) of ethyl acetate extraction ofreaction mixtures contained 100 μM resveratrol, 300 μM DMAPP, 10 mMMgCl₂, 5 mM DTT and 30 microsomal fraction in a pH 9.0 Tris-HCl bufferfor 40 min.

(C) HPLC-PAD-ESI-MS³ analysis of prenylated product, arachidin-2.

FIG. 45 shows the Comparison of AhR4DT-1 and AhRPT-9i2.

(A) Alignment of AhR4DT-1 with AhRPT-9i2 was performed using ClustalX.

(B) Potential transmembrane domains of AhR4DT-1 and AhRPT-9i2 werepredicted by TMHMM.

(C) Resveratrol prenylation activity of AhR4DT-1 and AhRPT-9i2 wereanalyzed by using HPLC.

FIG. 46 shows a comparison of AhR3′DT-1, AhRPT-10a4 and AhRPT-10d4.

(A) Alignment of AhR3′DT-1, AhRPT-10a4 and AhRPT-10d4 was performedusing ClustalX.

(B) Potential transmembrane domains of AhR3′DT-1, AhRPT-10a4 andAhRPT-10d4 were predicted by TMHMM.

(C) Resveratrol prenylation activity of AhR3′DT-1, AhRPT-10a4 andAhRPT-10d4 were analyzed by using HPLC.

FIG. 47 shows a comparison of AhR3′DT-2 and AhR3′DT-3.

(A) Alignment of AhR3′DT-2 with AhR3′DT-3 was performed using ClustalX.

(B) Potential transmembrane domains of AhR3′DT-2 and AhR3′DT-3 werepredicted by TMHMM.

(C) Resveratrol prenylation activity of AhR3′DT-2 and AhR3′DT-3 wereanalyzed by using HPLC.

FIG. 48 shows a structural analysis of AhR3′DT-4.

(A) Primary structure of AhR3′DT-4.

(B) Potential transmembrane domains of AhR3′DT-4 was predicted by TMHMM.

(C) Resveratrol prenylation activity of AhR3′DT-4 was analyzed by usingHPLC.

FIG. 49 shows a temperature dependency of AhR4DT-1 and AhR3′DT-1activity. AhR4DT-1 (A) and AhR3′DT-1 (B) activities were measured atvarious temperature (20, 25, 28, 30, 37, 40 and 50° C.) in 100 mMTris-HCl buffer (pH 9.0) for 40 mins.

FIG. 50 shows the incubation time and amounts of microsomal fractiondependency of AhR4DT-1 and AhR3′DT-1 activity.

(A) Concentrations of generated prenylated resveratrol from AhR4DT-1 andAhR3′DT-1 reaction mixtures with varying incubation times (30, 60, 90and 120 min) were quantified by HPLC.

(B) Concentrations of generated prenylated resveratrol from AhR4DT-1 andAhR3′DT-1 reaction mixtures with varying amounts of microsomal fraction(25, 50, 75 and 100 μg) were quantified by HPLC.

FIG. 51 shows a pH dependency of AhR4DT-1 and AhR3′DT-1 activity.AhR4DT-1 (A) and AhR3′DT-1 (B) activities at various pH values weremeasured in three different buffers: 100 mM Tris-HCl buffer at pH 7.0,8.0, 8.6 and 9.0; 100 mM Glycine-NaOH buffer at pH 8.6, 9.0, 9.4, 10.0and 10.6; and 100 mM NaHCO₃—Na₂CO₃ buffer at pH 9.2, 9.7, 10.2 and 10.7.All the reactions were performed at 28° C. for 40 min.

FIG. 52 shows a divalent cation dependency of AhR4DT-1 and AhR3′DT-1activity. AhR4DT-1 (A) and AhR3′DT-1 (B) activity with various divalentcation were measured with 10 mM MnCl₂, FeCl₂, CaCl₂, CoCl₂, ZnCl₂,NiCl₂, or CuCl₂ and the enzyme activity was compared with the reactionof 10 mM MgCl₂. Reactions without divalent cation and 10 mM EDTA insteadof MgCl₂ were used as controls. All the reactions were performed in 100mM Tris-HCl buffer (pH 9.0) at 28° C. for 40 min. (t.a., trace amount(<0.5%), n.d., Not detected.). Means and the standard deviation (errorbars) were calculated from three replicates.

FIG. 53 shows the kinetic values of AhR4DT-1 and AhR3′DT-1. Dependencyof AhR4DT-1 (left) and AhR3′DT-1 (right) on the concentration ofresveratrol (A&D), piceatannol (B&E) and DMAPP (C&F) measured with amicrosomal fraction from leaves of Nicotiana benthamiana. The apparentK_(m) and V_(max) values for resveratrol and piceatannol were determinedwith varying concentrations (10˜640 μM) using 640 μM DMAPP as prenyldonor, while that for DMAPP were determined with varying concentrations(10˜640 μM) using 640 μM resveratrol as prenyl acceptor. All the valueswere calculated from nonlinear regression analysis with Michaelis-Mentenequation by Graphpad Prism 6 software. Means and the standard deviation(error bars) were calculated from three replicates.

FIG. 54 shows the prenylation of piceatannol by AhR4DT-1.

Substrate specificity of AhR4DT-1 in microsomal fraction of Nicotianabenthamiana leaf after vacuum infiltration with Agrobacteriumtumefaciens LBA4404 harboring pBIB-Kan-AhR4DT-1 was analyzed by HPLC andLC-MS^(n).

(A) Chemical structures of resveratrol and its prenylated product.

(B) HPLC chromatograms (UV 320 nm) of ethyl acetate extraction ofreaction mixtures contained 100 μM piceatannol, 300 μM DMAPP, 10 mMMgCl₂, 5 mM DTT and 30 microsomal fraction in a pH 9.0 Tris-HCl bufferfor 40 min.

(C) HPLC-PAD-ESI-MS³ analysis of prenylated product, arachidin-5.

FIG. 55 shows the prenylation of pinosylvin by AhR4DT-1.

Substrate specificity of AhR4DT-1 in microsomal fraction of Nicotianabenthamiana leaf after vacuum infiltration with Agrobacteriumtumefaciens LBA4404 harboring pBIB-Kan-AhR4DT-1 was analyzed by HPLC andLC-MS^(n).

(A) Chemical structures of resveratrol and its prenylated product.

(B) HPLC chromatograms (UV 320 nm) of ethyl acetate extraction ofreaction mixtures contained 100 μM pinosylvin, 300 μM DMAPP, 10 mMMgCl₂, 5 mM DTT and 30 μg microsomal fraction in a pH 9.0 Tris-HClbuffer for 40 min.

(C) HPLC-PAD-ESI-MS³ analysis of prenylated product, chiricanine A.

FIG. 56 shows the prenylation of oxyresveratrol by AhR4DT-1.

Substrate specificity of AhR4DT-1 in microsomal fraction of Nicotianabenthamiana leaf after vacuum infiltration with Agrobacteriumtumefaciens LBA4404 harboring pBIB-Kan-AhR4DT-1 was analyzed by HPLC andLC-MS″.

(A) Chemical structures of resveratrol and its prenylated product.

(B) HPLC chromatograms (UV 320 nm) of ethyl acetate extraction ofreaction mixtures contained 100 μM oxyresveratrol, 300 μM DMAPP, 10 mMMgCl₂, 5 mM DTT and 30 μg microsomal fraction in a pH 9.0 Tris-HClbuffer for 40 min.

(C) HPLC-PAD-ESI-MS³ analysis of prenylated product.

FIG. 57 shows the prenylation of piceatannol by AhR3′DT-1.

Substrate specificity of AhR3′DT-1 in microsomal fraction of Nicotianabenthamiana leaf after vacuum infiltration with Agrobacteriumtumefaciens LBA4404 harboring pBIB-Kan-AhR3′DT-1 was analyzed by HPLCand LC-MS″.

(A) Chemical structures of resveratrol and its prenylated product,arachidin-2.

(B), HPLC chromatograms (UV 320 nm) of ethyl acetate extraction ofreaction mixtures contained 100 μM piceatannol, 300 μM DMAPP, 10 mMMgCl₂, 5 mM DTT and 30 microsomal fraction in a pH 9.0 Tris-HCl bufferfor 40 min.

(C) HPLC-PAD-ESI-MS³ analysis of prenylated product.

FIG. 58 shows the prenylation of oxyresveratrol by AhR3′DT-1.

Substrate specificity of AhR3′DT-1 in microsomal fraction of Nicotianabenthamiana leaf after vacuum infiltration with Agrobacteriumtumefaciens LBA4404 harboring pBM-Kan-AhR3′DT-1 was analyzed by HPLC andLC-MS″.

(A) Chemical structures of resveratrol and its prenylated product,arachidin-2.

(B) HPLC chromatograms (UV 320 nm) of ethyl acetate extraction ofreaction mixtures contained 100 μM oxyresveratrol, 300 μM DMAPP, 10 mMMgCl₂, 5 mM DTT and 30 microsomal fraction in a pH 9.0 Tris-HCl bufferfor 40 min.

(C) HPLC-PAD-ESI-MS³ analysis of prenylated product.

FIGS. 59, 59A, 59B, 59C, 59D, 59E, 59F, and 59G show the cloningstrategy of binary vectors for peanut prenyltransferase genes screening.

FIGS. 60, 60A, 60B, 60C, 60D, 60E, 60F, and 60G show the cloningstrategy of binary vectors for subcellular localization of AhR4DT-1 orAhR3′DT-1.

FIGS. 61, 61A, 61B, 61C, 61D, 61E, 61F, and 61G show the cloningstrategy of binary vectors containing GFP for subcellular localizationcontrol.

FIG. 62 shows the primer efficiencies for real-time qPCR.

The efficiency of AhR4DT-1 (A) was tested in 5× serial dilutions ofpeanut hairy root cDNA (range of 2 to 0.00032 ng). The efficiencies ofAhR3′DT-1 (B) was calculated in the range of 10 to 0.0064 ng cDNA, andthat of ATC7 (C) and EFα1 (D) were performed with 10 to 0.00032 ng cDNA.

FIG. 63 shows a table of Prenyltransferase transcripts described in thisapplication. Activities of expressed protein products and loci of bestalignment in diploid Arachis reference genomes are shown.

FIG. 64 shows a table of AhR4DT-1 and AhR3′DT-1 activity from N.benthamiana leaves fractions. The preparation of fractions and theresveratrol prenyltransferase assay are described in “Materials andMethods.” Values are the mean±standard deviation for three replicates.

FIG. 65 shows a table of comparison of kinetic values of AhR4DT-1,AhR3′DT-1 and prenyltransferase activity identified from peanut hairyroot. The apparent K_(m) and V_(max) values of AhR4DT-1 and AhR3′DT-1for resveratrol, piceatannol and DMAPP were measured using themicrosomal fraction of N. benthamiana leaves transiently expressingthese two enzymes. Values are the mean±standard deviation of threereplicates. The apparent K_(m) and V_(max) values of prenyltransferaseactivity identified from the microsomal fraction of peanut hairy rootfor resveratrol and DMAPP were previously reported by Yang et al.(2016).

FIGS. 66A-66B show a list of primers used in this study. The restrictionsite on each primer is underlined. (SEQ ID NOs:21-51)

FIG. 67 shows substrates used for specificity assay and the prenylatedproducts from reaction mixtures catalyzed by AhR4DT-1 or AhR3′DT-1.Analysis was done by HPLC-PDA-electrospray ionization-MS3.

FIG. 68 shows a list of plasmids used in this study. For details of thebinary vectors, see Materials and Methods.

FIGS. 69A-69B show accession numbers of proteins used for phylogeneticanalysis.

FIG. 70 shows a primer design for analysis of transgenic plantsexpressing peanut stilbenoid-specific prenyltransferases (AhR4DT-1 andAhR3′DT-1). Targeting position of primers, SubLoc-F-pBIBKan andSubLoc-R-pBIBKan on (A) pBIBKan, (B) pBIB-Kan-AhR4DT-1 and (C)pBIB-Kan-AhR3′DT-1.

FIG. 71 shows a characterization of transgenic tobacco plants expressingpeanut stilbenoid prenyltransferase AhR4DT-1 gene.

(A) AhR4DT-1 from peanut catalyzes the 4-C prenylation of resveratrol.

(B) PCR Analyses of transgenic tobacco plants expressing AhR4DT-1 gene.Genomic DNA was isolated from transgenic lines 1, 2, 3, 4, 5 and 6.Plasmid pBIB-Kan-AhR4DT-1 was used as positive control (+). Genomic DNAsof the transgenic plant transformed with pBIB-Kan empty vector and wildtype plant were used as empty vector control (−) and negative control,respectively. Positive amplicon (2673 bp); negative amplicon (523 bp).

(C) Enzymatic characterization of AhR4DT-1 activity in the leaf oftransgenic tobacco plants. HPLC chromatograms of ethyl acetate extractof 1 mL reaction mixture of 100 μM resveratrol, 300 μM DMAPP, 10 mMMgCl₂ and 5 mM DTT incubated with 50 μl of crude protein (approximately75 μg) from the leaf of transgenic tobacco plants in a pH 9.0 Tris-HClbuffer for 40 min. The crude protein from the leaf of transgenic planttransformed with pBIB-Kan vector was used as empty vector control.

FIG. 72 shows a characterization of transgenic tobacco plants expressingpeanut stilbenoid prenyltransferase AhR3′DT-1 gene.

(A) AhR3′DT-1 from peanut catalyzes the 3′-C prenylation of resveratrol.

(B) PCR Analyses of transgenic tobacco plants expressing AhR3′DT-1 gene.Genomic DNA was isolated from transgenic lines 1, 3, 4, 5, 6, 7, 8, 10,12 and 14. Plasmid pBIB-Kan-AhR3′DT-1 was used as positive control (+).Genomic DNAs of the transgenic plant transformed with pBIB-Kan vectorwas used as empty vector control (−). Positive amplicon (2619 bp);negative amplicon (523 bp).

(C) Enzymatic characterization of AhR3′DT-1 activity in the leaf oftransgenic tobacco lines. HPLC chromatograms of ethyl acetate extract of1 mL reaction mixture of 200 μM resveratrol, 300 μM DMAPP, 10 mM MgCl₂and 5 mM DTT incubated with 50 μl (approximately 75 μs) of crude proteinfrom the leaf of transgenic tobacco lines in a pH 9.0 Tris-HCl bufferfor 120 min. The crude protein from the leaf of transgenic planttransformed with pBIB-Kan vector was used as empty vector control.

FIG. 73 shows a phenotype of transgenic hairy roots of tobaccoexpressing peanut stilb enoid prenyltransferase AhR3′DT-1.

From left to right, hairy root lines developed from wild type of tobacco(WT), transgenic tobacco transformed with pBIB-Kan vector(pBIBKan—control), and AhR3′DT-1-expressing line 12 (Line 1 and Line 2).Hairy roots were developed via Agrobacterium rhizogenes-mediatedtransformation.

DETAILED DESCRIPTION

A substantial part of non-host defense responses in many plants is thepathogen-induced production of secondary metabolites, generally termedphytoalexins, that locally restrict disease progression due tobioactivities toxic to the pathogen (reviewed in Ahuj a et al., 2012).Peanut or groundnut (Arachis hypogaea) tissues mount a defense againstinfection by the soil fungus Aspergillus flavus and other pathogens byoverproducing stilbene derivatives around sites of wounding and elicitorperception (Sobolev, 2013). Resveratrol (3,5,4′-trihydroxy-stilbene),one of the most studied phytoalexin stilbenoids, has attracted greatattention because of its bioactive properties shown through in vitro andin vivo assays to benefit human health. These include anti-inflammatory(Das and Das, 2007) and antioxidant properties, as well as antitumor andfavorable cardiovascular effects (Gambini et al., 2015). However, thelimited oral bioavailability of resveratrol due to its rapid absorptionand metabolism restricts the future of this potentially valuable drug inclinical trials (Tome-Carneiro et al., 2013; Gambini et al., 2015).

Prenylated stilbenoids naturally produced as phytoalexins in the peanutplant possess one or two isoprenyl moieties bound to the aromatic ringof the stilbene molecule (FIG. 1). When compared to resveratrol, thesecompounds exhibit similar or enhanced bioactivity in in vitroexperiments. For instance, arachidin-1 and resveratrol showed similaranti-inflammatory activity in lipid polysaccharide-treated RAW 264.7macrophages and this correlated with the inhibition of prostaglandin E₂production (Chang et al., 2006; Djoko et al., 2007). Arachidin-1,arachidin-2 and arachidin-3, also applied to macrophages, were moreeffective than resveratrol in inhibiting inducible nitric oxideproduction (Sobolev et al., 2011). In other antioxidant activity assays,arachidin-1 inhibited lipid oxidation more effectively than resveratrol(Abbott et al., 2010), and arachidin-2 and arachidin-3 showed greaterpotency over resveratrol in inhibiting production of intracellularreactive oxygen species (Sobolev et al., 2011). Arachidin-1 furthershowed higher cytotoxicity than resveratrol to leukemia HL-60 cells(Huang et al., 2010) and other cancer cells (SK-MEL, KB, BT-549, andSK-OV-3) (Sobolev et al., 2011). Interestingly, arachidin-1 andarachidin-3 were shown to bind to human cannabinoid receptors 2(hCBR2s), while the affinity of their non-prenylated analogous,piceatannol and resveratrol for hCB2Rs was 5- to 10-fold lower.Molecular modeling studies with hCBR2s indicated that the prenyl moietyof the arachidins improved the binding affinity to the receptors (Brentset al., 2012).

In addition to the above-mentioned stilbenoids (arachidin-1, arachidin-2and arachidin-3), more than 20 other prenylated stilbenoids have beendescribed in peanut tissues (Sobolev et al., 2006; Wu et al., 2011;Sobolev, 2013; Sobolev et al., 2016). The biosynthesis of stilbenoidsderives from both the phenylpropanoid and acetate pathways. These mergeto produce resveratrol by the action of resveratrol synthase whichcatalyzes the cyclization of 4-coumaroyl-CoA and malonyl-CoA (Schoppnerand Kindl, 1984). The prenylation step, in which either of two prenylpatterns (3,3-dimethylallyl or 3-methyl-but-1-enyl) are introduced tovarious positions of the stilbene backbone (FIG. 1), along with theoxidation, methylation and cyclization steps plays a major role in thediversification of peanut prenylated stilbenoids. Although the enzymesinvolved in resveratrol biosynthesis have been elucidated (Chong et al.,2009), the enzymes involved in the prenylation steps of resveratrol orany other stilbenoid have not been described.

For other prenylated aromatic compounds, a prenyltransferase was foundto be the critical activity for coupling the aromatic compoundbiosynthesis and terpenoid biosynthesis, the latter leading to theformation of the prenyl unit (Yazaki et al., 2009). Two pathways areknown for the biosynthesis of prenylated compounds in plants, themevalonic acid (MVA) pathway in the cytosol and the2-C-methyl-D-erythritol-4-phosphate (MEP) pathway in the plastid (Lohret al., 2012). Many studies have shown that dimethylallyl pyrophosphate(DMAPP) derived from the MEP pathway is used as prenyl donor to formprenylated flavonoids or prenylated isoflavonoids in the plastid(Yamamoto et al., 2000; Yazaki et al., 2009). To determine thebiosynthetic origin of these terpenoids, distinct metabolic inhibitorswere applied to inhibit the key rate limiting enzymes involved in eitherthe MVA or MEP pathway. For instance, mevastatin, an inhibitor of3-hydroxy-3-methylglutaryl-coenzyme A reductase involved in the MVApathway was used in hairy root cultures of ginseng to study thebiosynthesis of ginsenosides (Zhao et al., 2014); while clomazone, aherbicide that inhibits 1-deoxy-D-xylulose-5-phosphate synthase (DXS)during early steps of DMAPP biosynthesis in the plastid was used toinvestigate the synthesis of monoterpenes in Catharanthus roseus (Han etal., 2013).

In order to elucidate the biosynthesis of peanut prenylated stilbenoids,we established hairy root cultures of peanut (Condon et al., 2010) andrecently demonstrated that a sustainable production of the prenylatedstilbenoids arachidin-1 and arachidin-3 can be achieved uponco-treatment of these cultures with methyl jasmonate (MeJA) andcyclodextrin (CD) (Yang et al., 2015a). In the current study, we tookadvantage of this bioproduction system and produced arachidin-2 and anew prenylated stilbenoid, named arachidin-5, as the prenylated productsof the hairy root microsomal fraction using resveratrol and piceatannolas substrates, respectively. To determine the biosynthetic origin of theprenyl moiety of these prenylated stilbenoids, two metabolic inhibitors,mevastatin and clomazone were selected and applied to peanut hairy rootcultures co-treated with MeJA and CD as elicitors. In the process, weidentified and characterized a resveratrol 4-dimethylallyltransferase(AhR4DT) from the microsomal fraction of elicited peanut hairy roots. Toour knowledge, this enzyme is the first stilbenoid-specificprenyltransferase that prenylates resveratrol and other specificstilbenoids at the 4-C position of the aromatic ring (FIG. 1).

Results

Purification and Structural Elucidation of a New Prenylated Stilbenoidfrom Peanut Hairy Root Culture

Hairy roots of peanut have the capability to produce and secreteresveratrol (3,5,4′-trihydroxy-trans-stilbene), piceatannol(3,5,3′,4′-tetrahydroxy-trans-stilbene) and their prenylated analogs,arachidin-3 and arachidin-1, in the medium upon co-treatment with MeJAand CD as elicitors (Yang et al., 2015a). In addition, in the currentstudy we also identified arachidin-2 in the ethyl acetate extract of theculture medium by comparing with the HPLC retention time, characteristicUV spectrum (λ_(max)323 mn) (FIG. 2) and mass spectrometric analysis ofthe purified arachidin-2 isolated from fungus-infected peanut seeds. Thestructure of arachidin-2 purified from peanut hairy root culture wassubsequently confirmed by ¹H- and ¹³C NMR spectra (data not shown)

A new prenylated stilbenoid with λ_(max) 327 nm in the HPLC mobile phasewas also isolated from the peanut hairy root culture medium (FIG. 2). Wenamed it arachidin-5. Mass spectrometry analysis (Table 1 shown in FIG.7) of arachidin-5 (m/z 313 [M+H]⁺) gave a main fragment with a m/z 257[M+H-56]⁺ in MS² which suggested the presence of a prenyl moiety.Arachidin-5 and arachidin-1 share very similar MS, MS² and MS³ spectra(Table 1 shown in FIG. 7) indicating that the structural differencebetween these two compounds might be only in the position of olefinicbond on their prenylated moieties.

The structure of the prenylated moiety on the arachidin-5 was furtherdetermined ¹H- and ¹³C-NMR The presence of a five-carbon unit of the3,3-dimethyiallyl structure was evident from the peak at 5.30 ppm (t,7.1 Hz) that is coupled to the peak at 3.36 ppm (d, 7.1), as well as thepeaks at 1.77 and 1.65 ppm for the two methyl groups (H-5″ nd H-4″,respectively) (FIG. 17). These proton resonances are the same as thosepublished for arachidin-2 (Park et 2011). The 3,3-dimethylallylstructure is also supported by the presence of a quaternary carbon peakat 130.9 (C-3″) in the ¹³C-NMR spectrum, as well as peaks at 124.4(C-2″), 26.0 (C-4″), 23.1 (C-1″) and 17.9 (C-5″) (FIG. 18). Theseresonances are in agreement with published resonances for the isoprenetail of arachidin-2 (Park et al., 2011). The NMR results showed thatarachidin-5 has the same 3,3-dimethylallyl moiety as arachidin-2 but anadditional hydroxyl group at the 3′ position, while arachidin-1 andarachidin-3 have the 3-methyl-but-1-enyl moieties instead.

In addition to arachidin-5, arachidin-1, arachidin-2 and arachidin-3,HPLC analysis of the culture medium showed the presence of 2 othercompounds with similar characteristic of arachidin-5 and arachidin-2based on UV and MS, MS² and MS³ spectra (FIG. 2 and Table 1 shown inFIG. 7). These compounds were later designated as arachidin-5 derivativeand arachidin-2 derivative respectively.

Effects of Metabolic Inhibitors on Yield of Prenylated Stilbenoids inPeanut Hairy Root Culture

As expected for peanut phytoalexins, non-prenylated and prenylatedstilbenoids were only present in the peanut hairy root cultures afterelicitor treatment (Medina-Bolivar et al., 2007; Yang et al., 2015a). Tostudy the metabolic origin of the prenyl moiety of the prenylatedstilbenods, mevastatin or clomazone was added to 9-day peanut hairy rootcultures co-treated with 100 μM MeJA and 9 g/L CD. In preliminaryexperiments we found that mevastatin at 1, 10, or 100 μM did not affectthe levels of prenylated stilbenoids (FIG. 19). On the other hand, theeffects on the accumulation of resveratrol and inhibition of prenylatedstilbenoids accumulation were increased with the concentration ofclomazone (1 μM to 100 μM) (FIG. 20). Hence, to quantify these effects,we determined yields of resveratrol and prenylated stilbenoids after 48h and 72 h of elicitor treatment together with mevastatin (10 μM), orclomazone (10 μM and 100 μM). The yields of stilbenoids were expressedin micromoles to assess the molar contribution of resveratrol as aprecursor of the prenylated stilbenoids.

Mevastatin had no significant effect on the yields of resveratrol,piceatannol and prenylated stilbenoids with the exception of arachidin-5and arachidin-1, which showed a 27% and 41% increase in yield,respectively, after a 72-h treatment (FIG. 3). During the 24-h intervalbetween 48 and 72-h treatments, non-inhibitor and mevastatin treatedgroups had significant increases in the yields of arachidin-5,arachidin-1, arachidin-2, and arachidin-3 indicating that mevastatin didnot inhibit the accumulation of these prenylated stilbenoids (FIG. 3).

With an inherent limited production (about hundred fold lower than thatof resveratrol) upon elicitor treatment, piceatannol concentrationincreased only slightly in the 100 μM clomazone group after 72 h oftreatment (FIG. 3). However, in both 10 μM and 100 μM clomazone treatedgroups, there were significantly higher yields of resveratrol andsignificantly lower yields of prenylated stilbenoids when compared tothe non-inhibitor group. In particular, the accumulation of arachidin-5and arachidin-1 was almost completely inhibited and only trace amountswere observed in the 48-hour treated groups containing 100 μM clomazone(FIG. 3). There were no significant increases in the levels of allprenylated stilbenoids during the 24-h interval between the 48- and 72-htime points indicating that clomazone has an inhibitory effect on theaccumulation of prenylated stilbenoids in peanut hairy root cultures.Since each of the inhibitors was applied to 9-day-old hairy rootcultures together with elicitors, DMAPP or IPP might have been alreadysynthesized and stored in the tissue prior to the inhibition. Thus, evenif the biosynthesis of DMAPP and IPP was blocked when clomazone wasadded into the medium, small amounts of arachidin-2 and arachidin-3could be detected in the 48- and 72-h clomazone treated samples (FIG.3). Moreover, when different concentrations of clomazone were applied tothe peanut hairy root culture without elicitors, none of thesestilbenoids was detected in the ethyl acetate extracts of the culturemedium suggesting that clomazone was not able to induce the productionof stilbenoids by itself (FIG. 21). However, in the co-treated group of100 clomazone with elicitors, the yield of resveratrol reached up to830.73±25.83 μM after 72 h which was 6.3 fold higher than that in thenon-inhibitor group. The micromolar increase of resveratrol was about1.54 fold greater than the overall decrease in the accumulation ofarachidin-5, arachidin-1, arachidin-2, and arachidin-3, suggesting thatthese prenylated stilbenoids may have been derived from resveratrol.

Degradation of Exogenous Resveratrol in Peanut Hairy Roots

Upon co-treatment of peanut hairy root cultures with MeJA and CD aselicitors, most of the total resveratrol and prenylated stilbenoidsproduced were secreted into the culture medium and only trace amountswere found in the ethyl acetate extracts of the root tissue (FIG. 22).Based on previous observations regarding loss of resveratrolaccumulation in medium of non-elicited peanut hairy root cultures (Yanget al., 2015a), we speculated that resveratrol could be taken up by thehairy roots and metabolized by one or multiple enzymatic mechanisms inthe roots.

To confirm this hypothesis, 1 mM resveratrol was co-incubated with acrude cell-free extract from non-elicited peanut hairy roots. After30-min incubation, the concentration of resveratrol declined to 53% ofthat in the control group co-incubated with heat-denatured crudecell-free extract. Meanwhile, piceatannol and other unidentifiedcompound with λ_(max) 327 nm referred to as a resveratrol derivative,were detected as reaction products (FIG. 4a and FIG. 4d ). In fact,according to our preliminary experiments, not only was resveratrolmetabolized, but the accumulation of prenylated stilbenoids likearachidin-2 was likewise affected. Consequently, after 120-minincubation, neither resveratrol nor its prenylation product, arachidin-2was detected in the ethyl acetate extracts of the reaction mixtures(FIG. 23). To prevent the oxidation of resveratrol, dithiothreitol (DTT)as reducing agent was added to the reaction mixture of the crudecell-free extract from non-elicited peanut hairy root using 1 mMresveratrol as substrate. After 120-min co-incubation with 5 mM DTT,more than 95% of the amount of resveratrol remained and no piceatannolwas detected in the mixture (FIG. 4c ), suggesting that DTT was able toprevent the oxidation of resveratrol.

Detection of Resveratrol Prenyltransferase (AhR4DT) Activity in PeanutHairy Roots

All flavonoid-specific prenyltransferases reported to date from otherlegume plants have been localized to plastid membranes, and resultsdescribed above indicate that the accumulation of prenylated stilbenoidsin our hairy root culture system can be blocked in the presence ofplastid MEP pathway inhibitor. We therefore hypothesized that thestilbenoid-specific prenyltransferase(s) in peanut might also bemembrane localized and use DMAPP or IPP originating from the MEP pathwayas the prenyl donor. Furthermore, our clomazone inhibitor experimentssuggested resveratrol as a prenyl acceptor and a precursor of prenylatedstilbenoids. We therefore prepared a microsomal fraction usingultracentrifugation from 48-h elicited peanut hairy root treated withresveratrol, DMAPP, along with DTT to reduce the degradation ofresveratrol and its prenylated product. Two enzymatic products weresynthesized in the reaction mixture; the predominant one was identifiedas arachidin-2 by comparison of its retention time, UV spectrum, massspectra and fragmentation patterns obtained by tandem mass spectrometry(MS² and MS³), each of which were identical to that of arachidin-2purified from peanut hairy root culture (FIG. 5b ). Another product(FIG. 5b ) shared identical retention time, UV spectrum, mass spectra,MS² and MS³ with that of unidentified compound found in peanut hairyroot culture (Table 1 shown in FIG. 7). Due to the presence in theenzymatic reaction with 100 arachidin-2 and crude cell-free extract(FIG. 24), this product is considered as an enzymatic derivative ofarachidin-2. None of the prenylated products were detected in the sampleincubated with the microsomal fraction from non-elicited hairy roottissue (FIG. 28), indicating the inducibility of this resveratrolprenyltransferase in peanut hairy roots. Moreover, the specific activityof resveratrol prenyltransferase in the microsomal fraction(421.16±16.25 pkat·mg⁻¹ of protein, based on the production ofarachidin-2) was 9.3-fold higher than that in the crude cell-freeextracts, suggesting that resveratrol prenyltransferase in peanut wasbound to the membrane fraction of the root cells (Table 2 shown in FIG.8).

Biochemical Characterization of Resveratrol Prenyltransferase AhR4DT

In the resveratrol prenyltransferase reaction, the accumulation of theprenylated product arachidin-2 followed a linear relationship with inputbetween 30 μg and 120 μg of microsomal protein. The reaction was alsolinear over the 120 min incubation time (FIG. 25). The optimum pH forthis prenyltransferase was 9.2 in a Tris-HCl buffer (FIG. 26), which wasclose to the optimum pH values of 9-10 for flavonoid specificprenyltransferases (Yamamoto et al., 2000). Divalent cations wereabsolutely required for resveratrol prenyltransferase activity and Mg²⁺was the most effective these (100%), followed by Mn²⁺ (71.8%), Fe²⁺(14.6%) and Ca²⁺ (0.9%) (FIG. 27). No prenyltransferase activity wasdetected when a divalent cation was absent from the reaction (FIG. 28).Isoprenoid precursor isopentenyl diphosphate (IPP) was tested as aprenyl donor for the prenyltransferase with resveratrol as the prenylacceptor and no activity was detected in the assay (FIG. 28). Theapparent k_(m) values for resveratrol and DMAPP were calculated as111.1±40.44 μM and 91.89±7.032 respectively (FIG. 29).

Substrate Specificity of Resveratrol Prenyltransferase AhR4DT

To analyze the substrate specificity of the resveratrolprenyltransferase from peanut hairy root, stilbenoids (piceatannol,pinosylvin, pterostilbene and oxyresveratrol), flavanone (naringenin),flavone (apigenin) and isoflavone (genistein) were incubated with amicrosomal fraction using DMAPP as a prenyl donor. When piceatannol wasused as a prenyl acceptor, the microsomal fraction catalyzed thesynthesis of arachidin-5 as the predominant prenylated product (FIG.30). Similar to the arachidin-2 derivative, another product consideredas an enzymatic derivative of arachidin-5, was also detected in thereaction (FIG. 31). In addition, the microsomal fraction catalyzedpinosylvin into chiricanine A, confirmed by comparison of its retentiontime, UV spectrum, mass spectra and fragmentation patterns with those ofchiricanine-A standard purified from fungus-challenged peanut seeds(FIG. 6 and FIG. 30). Using this microsomal fraction, an isoprene unitwas also transferred to oxyresveratrol. Mass spectroscopic analysis ofits reaction product (m/z 313 [M+H]⁺) showed a main fragment with a m/z257 [M+H-56]⁺ in MS² which suggested the presence of a prenyl moiety(FIG. 32). However, the position of the prenyl moiety on this prenylatedoxyresveratrol remains undetermined due to the insufficient amount ofcompound for further structure elucidation. Interestingly, pterostilbenewhich has methoxy groups at C-3 and C-5 positions, respectively, did notproduce any product within the microsomal fraction (FIG. 6 and FIG. 30),suggesting that the additional methyl group on its structure might blockprenylation and that either or both of the hydroxyl groups at C-3 andC-5 position might be required for the prenylation reaction.Furthermore, neither prenylated flavanone, prenylated flavone norprenylated isoflavone was detected in the reaction mixtures (FIG. 6 andFIG. 30), indicating that this peanut prenyltransferase may be astilbenoid-specific prenyltransferase.

Discussion

AhR4DT, a Membrane-Bound Prenyltransferase Specific for Stilbenoids fromPeanut Hairy Root

As the limiting enzyme involved in the biosynthesis of prenylatedflavonoids, flavonoid prenyltansferases have been a focus of previousresearch particularly in plants of the Leguminosae family known toaccumulate these type of specialized metabolites. The first flavonoidprenyltansferase, SfN8DT, was identified in Sophora flavescens cellcultures by Sasaki et al. in 2008. Subsequently, SfG6DT and SfiLDT,which prenylate genistein to produce wighteone(6-dimethylallylgenistein) and isoliquiritigenin todimethyllylisoliquiritigenin respectively, were identified in the samespecies (Sasaki et al., 2011). While SfG6DT prenylates genistein on theA-ring of the isoflavone, LaPT1 isolated from another legume, whitelupin (Lupinus albus), catalyzes the prenylation of the B-ring ofgenistein and 2′-hydroxygenistein to form isowighteone(3′-dimethylallylgenistein) and luteone(2′-hydroxy-3′-dimethylallyl-genistein), respectively (Shen et al.,2012). As a crucial prenyltransferase involved in glyceollinbiosynthesis, GmG4DT was identified and characterized in soybean(Glycine max). This enzyme catalyzes the dimethylallylation of glycinolat position 4 to produce the precursor of the phytoalexin, glyceollin I(Akashi et al., 2008). More recently, GuA6DT, a flavoneprenyltransferase was identified in another legume species, liquorice(Glycyrrhiza uralensis) (Li et al., 2014).

In this study, we have described the first plant stilbenoidprenyltransferase activity, and named this enzyme as resveratrol4-dimethylallyltransferase (AhR4DT). AhR4DT catalyzes thedimethylallylation of resveratrol at C-4 and is derived from themicrosomal fraction of elicited peanut hairy roots. AhR4DT shows severalcommon features with other prenyltransferases, such as those describedabove. For instance, all prenylation activities mentioned above anddemonstrated here require a divalent cation as cofactor and a basicbuffer for optimal reaction rate (except for GmG4DT which reaction rateoptimum is pH 7.5 buffer for the reaction, (Akashi et al., 2008).Because the activity of AhR4DT was concentrated in the microsomalfraction and the accumulation of prenylated stilbenoid in peanut hairyroots was inhibited by clomazone, we suggest that this peanutresveratrol prenyltransferase is a membrane-bound protein located in theplastid. This further suggests that the DMAPP used in the prenylationreaction was derived from the MEP plastidic pathway. Our hypothesis isin agreement with the plastidic subcellular location of flavonoidprenyltransferases identified in other legume species.

During the biosynthesis of stilbenoids and flavonoids, both resveratrolsynthase and chalcone synthase use 4-coumaroyl-CoA as a substrate andperform three condensation reactions with malonyl-CoA to form a lineartetraketide, which is later folded into new ring structures. These twoenzymes are distinguished by a special property of stilbene synthases,which looses the terminal carboxyl group as CO₂, resulting in release of4 CO₂. In contrast, each reaction catalyzed by chalcone synthasereleases 3 CO₂ molecules. It has been suggested that stilbene synthasemay have developed from chalcone synthase via gene duplication andmutation rendering new and improved functions (Tropf et al., 1994).

Among the prenylation products of resveratrol, piceatannol, pinosylvin,and oxyresveratrol catalyzed by the microsomal fraction containingAhR4DT, only arachidin-2 and arachidin-5 were detected in the spentmedium of peanut hairy root cultures co-treated with MeJA and CD.Interestingly, oxyresveratrol and prenylated oxyresveratrol reported inthe bark of Artocarpus dadah (Su et al., 2002) along with pinosylvinisolated from pine hardwood, have not been identified in any peanuttissue, while the prenylated product of pinosylvin, chiricanine A thatwas first described in the roots of Lonchocarpus chiricanus (Ioset etal., 2001), has been described in peanut seeds after infection with thefungus Aspergillus flavus (Sobolev et al., 2009).

Biosynthesis of Prenylated Stilbenoids in Peanut Hairy Root Cultures

The use of abiotic elicitors to induce the biosynthesis of stilbenoid inhairy root cultures provides for an axenic sustainable and controlledproduction platform for these specialized metabolites which can beleveraged to study their biosynthetic pathway. The metabolic steps toproduce resveratrol from phenylalanine including phenylalanineammonia-lyase (PAL), cinnamate-4-hydroxylase (C4H), 4-coumarate:coenzymeA ligase (4CL), and resveratrol synthase (RS) have already beenelucidated (Watts et al., 2006). In the current study, a stilbenoidprenyltransferase, AhR4DT, which prenylates resveratrol to formarachidin-2 was characterized from the microsomal faction of elicitedpeanut hairy roots. Since resveratrol accumulated in the medium whileclomazone blocked the plastid MEP pathway in our inhibitor feedingexperiments, it may be that prenylated stilbenoids in addition toarachidin-2 also derive from resveratrol. A study of phytoalexinsinduced in peanut kernels by soil fungal exposures also suggests thatresveratrol might be the precursor for other prenylated stilbenoids inpeanut (Sobolev, 2008).

The main prenylated stilbenoids produced by peanut hairy root can becategorized into two groups according to the structure of their prenylside chains. One group includes arachidin-2 and arachidin-5 (identifiedin this study) having a 3,3-dimethylallyl moiety. This is the mostcommon type of prenylation found in prenylated stilbenoids from variousplant families. For instance, longistylines C, longistylines D andchiricanine A found in Lonchocarpus chiricanus (Leguminosae) (Ioset etal., 2001) also have the same dimethylallyl moiety. Artoindonesianin Nwith one dimethylallyl moiety and 4-dimethylallyl-oxystilbene werereported in Artocarpus integer (Moraceae) (Boonlaksiri et al., 2000) andA. gomezianus (Moraceae) (Hakim et al., 2002) respectively. Mappainfound in Macaranga mappa (Euphorbiaceae) (Van Der Kaaden et al., 2001)has one dimethylallyl moiety and one geranyl moiety, whileschweinfurthin C with two geranyl moieties was isolated from M.alnifolia (Euphorbiaceae) (Yoder et al., 2007). Moreover, thedimethylallyl moiety is also the most common prenylation pattern presentin prenylated flavonoids, which are mostly found in the followingfamilies: Cannabaceae, Guttiferae, Leguminosae, Moraceae, Rutaceae, andUmbelliferae. The longer form of the dimethylallyl moiety, geranyl andlavandulyl moiety, has also been reported in prenylated flavonoids(Botta et al., 2005; Yang et al., 2015b). During the biosynthesis ofthese prenylated stilbenoids and prenylated flavonoids,prenyltransferases are responsible for these dimethylallylation andgeranylation attaching DMAPP and GPP, respectively, to differentpositions of the stilbenoid and flavonoid skeletons.

One major group of prenylated stilbenoids in peanut hairy roots isrepresented by arachidin-1 and arachidin-3 which harbor a3-methyl-but-1-enyl moiety. When compared with the dimethylallylmoieties commonly present in other prenylated stilbenoids andflavonoids, this unique prenylated form has been reported in peanut andvery few other species. To our knowledge, the3-methyl-but-1-enyl-oxystilbene isolated from A. integer (Moraceae)(Boonlaksiri et al., 2000) is the only prenylated stilbenoid with thismoiety reported in a species other than peanut. Due to the difference inthe position of the olefinic bond on the prenylated moieties,3-methyl-but-1-enyl stilbenoids such arachidin-3 and arachidin-1 havehigher lipophilicity as evident from a later retention time inreverse-phase HPLC chromatograms when compared with their3,3-dimethylallyl analogs, arachidin-2 and arachidin-5 respectively. Themax of UV absorbance of 3-methyl-but-1-enyl stilbenoids has an apparentshift to 335˜340 nm when compared with that shown at 323˜327 nm for3,3-dimethylallyl stilbenoids (Table 1 shown in FIG. 7). However, theeffects of these two moieties on the bioactivity of peanut stilbenoidsis still unclear because arachidin-1 and arachidin-3 are notcommercially available limiting their studies and the relatively lowyields of arachidin-2 and arachidin-5 produced in peanut hairy roots.

The yields of arachidin-1 and arachidin-3 in our peanut hairy rootculture system were much higher than those of their dimethylallylanalogs, arachidin-2 and arachidin-5. As described in the activityassays above, both arachidin-2 and arachidin-5 were further modified toother derivatives, suggesting that dimethylallyl stilbenoids areimportant intermediates for the biosynthesis of other peanut prenylatedstilbenoids. As a substrate recognized by AhR4DT, piceatannol wasconsidered to be the putative precursor of arachidin-5 in peanut.However, the yield of piceatannol in our peanut hairy root culture isvery limited and far less than that of resveratrol. Even after blockingthe biosynthesis of the prenylated moiety by 100 μM clomazone, thecultures only produced 9.02 μM of piceatannol compared with 845.7 μM ofresveratrol (FIG. 3). These observations suggests that arachidin-5 andarachidin-1 might not be synthesized from piceatannol due to its limitedamounts, instead arachidin-1 may derive from arachidin-3, which isrelatively abundant in the medium, by hydroxylation at C-3′ position.

Metabolism of Prenylated Stilbenoids in Peanut Hairy Root

Under treatment with MeJA and CD, arachidin-1, arachidin-2, arachidin-3,and arachidin-5, are the major prenylated stilbenoids secreted andaccumulated in the spent culture together with resveratrol. When CD isnot added in the hairy root cultures, only limited amounts ofresveratrol and stilbenoids are detected in the medium. Theseobservations were reported previously when peanut hairy roots weretreated with NaOAc, H₂O₂ or MeJA alone as elicitors (Yang et al.,2015a). Importantly, when resveratrol was added in non-elicited peanuthairy root cultures, 47% and 97% reductions of the initial resveratrolconcentration in the culture medium were observed after 0.5 h and 1 hco-incubation, respectively (Yang et al., 2015a). In the current study,we confirmed that the degradation of extrinsic resveratrol in peanuthairy root culture occurs due to activities of the root tissue. Unlikeresveratrol synthase, which is induced and involved in stilbenoidbiosynthesis, the enzymes involved in the degradation of resveratrolappear to be constitutively expressed in non-elicited root tissue,leading to the dramatic decline of extrinsic resveratrol. Results fromexperiments described here suggest that peanut enzymes present initiatethis process by oxidizing resveratrol into piceatannol and then secondlyby converting this into other derivatives. This enzymatic degradationprocess may also apply to other prenylated stilbenoids, such asarachidin-2 (FIG. 23), and may explain the observation that only traceamounts of stilbenoids are detected in ethyl acetate extracts of roottissue (FIG. 22). The constitutive degradation of stilbenoids mayprovide the plant with the ability to manage potential toxic effect ofstilbenoids when they accumulate at high levels within the cell.Interestingly, other species such as grape accumulate resveratrol asglucosides (i.e. piceid). However, this conjugate was not identified inthe peanut hairy roots and has not been described in fungal elicitedpeanut kernels. Altogether, these findings suggest that peanut may haveevolved a distinct mechanism to metabolize resveratrol-type phytoalexinsafter they are produced.

Conclusion of Arachidin-5 Production

Harnessing the inducible bioproduction capabilities of the peanut hairyroot culture system, we have newly produced a prenylated stilbenoid,i.e., arachidin-5, and have demonstrated that the prenyl moiety onpeanut prenylated stilbenoids is derived from a plastidic biosynthesispathway. We have characterized for the first time a plant membrane-boundstilbenoid-specific prenyltransferase, i.e., AhR4DT, from the microsomalfraction of peanut hairy roots. The characteristics of AhR4DT provideimportant information for subsequent cloning and comprehensivedefinition of the prenyltransferase gene(s) of peanut. Moreover, we haveobserved the enzymatic degradation of exogenous resveratrol by peanuthairy root tissue, an observation that will lead to elucidation offurther mechanisms governing phytoalexin accumulation in plants.

Materials and Methods

Chemical Reagents

Authentic standards of resveratrol and piceatannol were obtained fromBiophysica and Axxora, respectively; arachidin-1, arachidin-2,arachidin-3, and arachidin-5 standards were purified from elicitedpeanut hairy root as described below. Pinosylvin, oxyresveratrol,pterostilbene, naringenin, apigenin, and genistein used in this studywere purchased from Sigma-Aldrich. Chiricanine-A was purified fromfungal-challenged peanut seeds as described below. DMAPP was obtainedfrom Isoprenoids, LC. Stock solutions of inhibitors, mevastatin (100 mM,Sigma-Aldrich) and clomazone (100 mM, Sigma-Aldrich), were prepared inethanol and stored at 4° C.

Isolation and Purification of Chiricanine-A from Fungal ChallengedPeanut Seeds

One kg of seeds of the 31-1314 peanut runner breeding line from theNational Peanut Research Laboratory (Dawson, Ga.), stored at 4° C. fortwo years after harvest, were allowed to imbibe distilled water for 18 hat 4° C. They were then chopped with a custom-made hand cutter into 3-6mm pieces, thoroughly washed with distilled water, blotted with a papertowel, air-dried to the condition where sliced peanuts did not leavewater spots on filter paper, and placed on aluminum trays so that thethickness of the layer did not exceed 1 cm. The trays were evenlysprayed with the fungal spores of Aspergillus caelatus NRRL 25528(10⁵/mL), placed into autoclave bags, and incubated at 30° C. for 96 h.The bags were opened every 24 h to allow fresh air to the peanuts andgrowing fungus.

The incubated peanut seeds were extracted with 3.0 L of MeOH overnightat room temperature without agitation. This procedure was repeated twomore times. The combined mixture was filtered through a paper filter ina Buchner type funnel under reduced pressure. The combined filtrateswere defatted three times with 0.5 L of n-hexane. The MeOH layer wasevaporated to dryness. The residue was redissolved in CHCl₃ and appliedto a chromatographic column (34 mm i.d.) packed with silica gel (silicagel 60, 0.063-0.200 mm; EM Science, Gibbstown, N.J.) to the height of400 mm. The column was subsequently eluted with 0.5 L of CHCl₃, 1.0 L ofEtOAc, 1.0 L of acetone, and 1.0 L of MeOH (all solvents were purchasedfrom Fisher). Eight fractions were collected from the column andanalyzed by HPLC. Fractions containing chiricanine A were combined,evaporated to dryness with a rotary evaporator, and subjected to furtherpurification on a similar silica gel column. The column was subsequentlyeluted with 0.4 L of CHCl₃, 1.5 L of CHCl₃/EtOAc (1:1), 1.3 L of EtOAc,and 1.0 L of acetone. Twenty four fractions were collected. Combinedfractions containing chiricanine A were evaporated to dryness on arotary evaporator, redissolved in MeOH, filtered, and subjected to finalpurification with a preparative 100 mm×19 mm i.d., 5 μm XTerra Prep RP18OBD HPLC column (Waters). The flow rate was 9.5 mL/min, and columntemperature was maintained at 40° C. The following mobile phase wasused: 73% CH₃CN, 3% of 1% HCOOH in H₂O, and 24% of H₂O. Pure fractionsof chiricanine A obtained from HPLC were evaporated with a rotaryevaporator to a point where almost all of the organic solvent wasremoved. Then the target compound was extracted four times with EtOAc(H₂O/EtOAc ratio 1:1, v/v). The combined EtOAc layers were evaporatednearly to dryness with a rotary evaporator. The residue was transferredinto a 15 mL vial with EtOAc and evaporated nearly to dryness with astream of N₂. The residue was redissolved in EtOAc, filtered, andtransferred into 4 mL vials and evaporated to dryness with a stream ofN₂. Then the vial was placed into a lyophilizer for 2 h at roomtemperature to remove traces of the solvents. Chiricanine A was obtainedas a slightly yellowish oil (6.5 mg).

Analyses of Stilbenoids from Peanut Hairy Root Cultures

Hairy root cultures of peanut (Arachis hypogaea) cv. Hull line 3established by the Medina-Bolivar laboratory (Condori et al., 2010) weremaintained in 50 mL modified Murashige and Skoog medium (MSV) (Condoriet al., 2010) with 3% sucrose in 250 ml flasks. In order to inducesynthesis and secretion of stilbenoids, the spent medium of nine-day-oldhairy root cultures was discarded and replaced with 50 mL fresh MSVmedium containing 3% sucrose with 100 μM methyl jasmonate (MeJA) and 9g/L methyl-β-cyclodextrin (CD; Cavasol™) as elicitors and incubated inthe dark at 28° C. for an additional 72 hours as previously described(Yang et al., 2015a). After elicitation period, one milliliter spentmedium was partitioned with ethyl acetate twice. The combined organicphase was evaporated under nitrogen gas and dissolved in methanol forsubsequent HPLC and mass spectrometry analyses.

Quantitative analysis of stilbenoids in peanut hairy root cultureextracts was performed in an UltiMate 3000 LC system (Dionex, ThermoScientific), equipped with a photodiode array detector. The separationwas performed on a SunFire™ C₁₈, 5 μm, 4.6×250 mm column (Waters) at 40°C. with a flow rate at 1.0 ml/min. The mobile phase consisted of 2%formic acid in water (A) and methanol (B). The column was initiallyequilibrated with 100% A for 1 min. Then a linear gradient was performedfrom 40% A and 60% B to 35% A and 65% B (1 to 20 min), followed by alinear gradient from 35% A and 65% B to 100% B (20 to 25 min). Then thecolumn was washed with 100% A for 5 min (25 to 30 min). Calibrationcurves of various stilbenoids were established using absorbance at 320nm for resveratrol, piceatannol, arachidin-2 and arachidin-5 and at 340nm for arachidin-1 and arachidin-3.

For LC-mass spectrometry qualitative analysis of stilbenoids, anUltiMate 3000 rapid separation LC system (Dionex, Thermo Scientific) wasused for the chromatographic separation. The separation method wassimilar to the HPLC conditions described above with the followingmodification, 0.02% formic acid in water was used instead 2% in themobile phase A. The LTQ XL™ linear ion trap mass spectrometer (ThermoScientific) with an electrospray ionization (ESI) source was used toobtain structural information of stilbenoids and followed the methoddescribed previously (Marsh et al., 2014). Briefly, all mass spectrawere performed in the positive ion mode with ion spray voltage at 4 kV,sheath gas (high purity nitrogen) at 45 arbitrary units (AU), auxiliarygas (high purity nitrogen) at 15 AU, capillary voltage at 9 V, capillarytemperature at 300° C., and tube lens offset at 45 V. Full mass scan wasrecorded in the range m/z 100-2000. Ultrahigh pure helium (He) was usedas the collision gas and 35% of collision energy was applied incollision-induced dissociation (CID). The data were recorded andanalyzed by Xcalibur software (Thermo Scientific).

Purification and Characterization of Arachidin-5 from Peanut Hairy RootCultures

For purification of arachidin-5 and other prenylated stilbeniods, oneliter of spent medium obtained from a pool of about 20 flasks of 72h-elicited peanut hairy root cultures was collected and extracted withan equal volume of ethyl acetate twice in a 2 L separatory funnel. Theorganic phase was recovered and dried in a rotavapor (Buchi) and thecrude extract (˜800 mg) was stored at 4° C. for subsequent highperformance counter current chromatography (HPCCC) fractionation. Atwo-phase HPCCC solvent system (hexane: ethyl acetate: methanol: water(4:5:3:3, v/v/v/v) was equilibrated at room temperature in a 2 Lseparatory funnel. The upper phase of this solvent mixture was used asstationary phase and the lower one was used as mobile phase forpreparative HPCCC (Dynamic Extractions) system. The multilayer coil wasfilled with the stationary phase at a flow rate of 8 mL/min whilespinning at 1600 rpm. Then the hydrodynamic equilibrium was establishedby pumping 6 mL/min of mobile phase into the column until a clear mobilephase was eluted at the outlet. Crude extract (300 mg) was dissolved in5 mL of the two-phase solvent and manually injected. The effluent wasmonitored at UV 340 nm. The fractions were collected every 30 s, driedin a SpeedVac, and analyzed by HPLC as described above. According to theHPLC profiles, the fractions collected between 27 to 32 min containedarachidin-1 and arachidin-5, whereas those collected between 53 to 62min contained arachidin-2 and arachidin-3. Fractions containingarachidin-1 and arachidin-5 were combined as one sample, whereas thosecontaining arachidin-2 and arachidin-3 were combined as a separatesample. Then, the organic solvent in the samples was removed usingrotavapor. The remaining aqueous mixtures containing the targetcompounds were extracted with ethyl acetate (1:1, v/v), evaporatednearly to dryness with a rotavapor and redissolved in methanol. Thenthese combined fractions were applied to TLC plates (TLC silica gel 60RP-18 F₂₅₄s, Millipore) and separated using as developing solvent asolvent system composed of methanol: water: acetic acid (15:45:40,v/v/v). After separation, the purified prenylated stilbenoids on theadsorbent were scraped off, re-dissolved in methanol and dried undernitrogen gas for subsequent ¹H and ¹³C NMR spectra analysis. In summary,4.6 mg of arachidin-5, 20.3 mg of arachidn-1, 5.2 mg of arachidin-2 and17.8 mg of arachidin-3 were purified from the peanut hairy root culturemedium using the HPCCC and preparative TLC method.

For NMR analysis of purified arachidin-5 and arachidin-2, ¹H NMR wasrecorded at 400 MHz, ¹³C NMR was recorded at 100 MHz in acetone-d₆ on aBruker AV-400 NMR spectrometer.

Inhibitor Feeding Experiment

Nine-day-old peanut hairy root cultures were used in the inhibitorfeeding experiment. Prior to treatment, the spent medium from eachculture was removed and replaced with 50 mL fresh MSV medium containing3% sucrose and 100 μM MeJA and 9 g/L CD as elicitors. Then, mevastatin(10 μM) or clomazone (10 μM and 100 μM) was applied to the culturemedium. For the control group, 50 μL of absolute ethanol (solvent ofmevastatin and clomazone) was added to the medium. All treatments wereperformed at 28° C. and continuous darkness with three biologicalreplicates per treatment. One milliliter of spent medium was collectedfrom each treatment at 48 h and 72h after treatment. The 1 mL mediumaliquots were extracted with 1 mL ethyl acetate, and then the organicphase was collected and dried under nitrogen gas and re-dissolved inmethanol for HPLC analysis as described above.

Enzyme Preparation

Enzyme solutions from peanut hairy roots for prenyltransferase assaywere prepared using nine-day-old roots elicited with 100 μM MeJA and 9g/L CD for 48 h. Ten grams (fresh weight) of elicited hairy root tissueswere grinded and homogenized in 20 mL of extraction buffer composed of100 mM Tris-HCL buffer (pH 7.6), 10 mM dithiothreitol (DTT) and 2.5%(w/v) polyvinylpyrrolidone (average mol wt 40,000; PVP-40, Sigma) usinga mortar and pestle. The homogenate was centrifuged at 12,000×g for 15min at 4° C. to remove the cell debris. Crude cell-free extracts wereobtained by passing 2.5 mL of the 12,000×g supernatant through a PD-10desalting column (GE Healthcare), using 100 mM Tris-HCL (pH 9.2)containing 10 mM DTT as equilibration buffer. About 13.5 mL of theremaining 12,000×g supernatant were subsequently centrifuged at156,000×g for 45 min at 4° C. The 156,000×g supernatant was collectedand cleaned up through a PD-10 desalting column (GE Healthcare)equilibrated with 100 mM Tris-HCl buffer (pH 9.2) containing 10 mM DTT.The microsomal pellet was resuspended in 100 mM Tris-HCl buffer (pH 9.2)containing 10 mM DTT, re-centrifugated (156,000×g for 45 min), andfinally resuspended in 1 mL of the same buffer for subsequent enzymereaction.

For the degradation of resveratrol assay, crude cell-free extract wasprepared from 12-day-old non-elicited peanut hairy roots following asimilar procedure to that described for elicited roots above except thatno DTT was included in the extraction and equilibration buffers.Protein Quantification

Protein contents in the various enzyme solutions were determined usingthe Coomassie protein assay (Thermo Scientific) using bovine serumalbumin as standard.

Degradation of Resveratrol Assay

The standard assay condition contained 1 mM resveratrol with 50 μg crudecell-free extract from 12-day-old non-elicited peanut hairy root in 500μL of 100 mM Tris-HCl buffer (pH 7.6). To study the effect of reducingagent on the degradation of resveratrol, additional 5 mM DTT was addedto the standard assay mixture. After 30 min incubation at 28° C., thereaction mixture was extracted with 500 μL ethyl acetate and the amountof resveratrol remaining in the mixture was analyzed by HPLC. In thecontrol group, crude cell-free extract was heated at 99° C. for 20 min.

Prenyltransferase Assay

The standard assay was performed in a total volume of 500 μL containing100 μM resveratrol, 300 μM DMAPP as a prenyl donor, 10 mM MgCl₂, 5 mMDTT and 30 μg microsomal fractions in 100 mM Tris-HCl buffer (pH 9.2).After 60 min incubation at 28° C., the reaction mixture was terminatedby adding 20 μL of 6 M HCl and extracted with 500 μL of ethyl acetate.The extracts were dried under nitrogen gas, dissolved in methanol andthe reaction product was quantified by HPLC analysis. The prenylationactivity for each reaction was quantified by the molar concentration ofgenerated prenylated product per second with one milligram microsomalfractions (kat/mg).

In the linearity study, the prenyltransferase activities under varyingincubation times (30, 60, 90 and 120 min) with 30 μg microsomal fractionand varying mount of microsomal faction (30, 60, 90 and 120 μg)incubated for 60 min were measured. For pH dependency study, theactivities of prenyltransferase were measured using 100 mM Tris-HClbuffer at pH 7.0, 8.0, 8.4, 8.6, 8.8, 9.0, 9.2, 9.4, 9.6, and 10.0. Fordivalent cation dependency study, 10 mM MnCl₂, FeCl₂, CaCl₂, CoCl₂,ZnCl₂, NiCl₂ or CuCl₂ was added to the reaction mixture instead of MgCl₂as described above, and the enzyme activity was compared with thatreaction containing MgCl₂. For kinetic studies, varying concentration(10, 20, 40, 80, 160, 320, and 640 μM) of resveratrol with a fixedconcentration of DMAPP (640 μM) and varying concentration (10, 20, 40,80, 160, 320, and 640 μM) of DMAPP with a fixed concentration ofresveratrol (640 μM) were incubated with microsomal fractions of peanuthairy root in a total volume of 250 μL at 28° C. for 60 min. Thesereactions were used to calculate V_(m) and K_(m) values using nonlinearregression analysis of Michaelis-Menten equation using Graphpad Prism 6software. For substrate specificity assay, 100 μM stilbenoids(resveratrol, piceatannol, pinosylvin, pterostilbene, andoxyresveratrol), flavanone (naringenin), flavone (apigenin), andisoflavone (genistein) with 300 μM DMAPP as a prenyl donor wereincubated with microsomal fractions of peanut hairy root in a totalvolume of 250 μL at 28° C. for 60 min. Arachidin-2 and arachidin-5purified from peanut hairy root culture and chiricanine-A purified fromfungus-challenged peanut seed were diluted to various concentration andcalibration curves of their absorbance at 320 nm were used forquantitative analysis.

Identification of Resveratrol Prenyltransferase Activities from the A.hypogaea Hairy Root Transcriptome

The first flavonoid-specific prenyltransferase, SfN8DT-1, was clonedfrom a cDNA (EST) library of Sophora flavescens cell cultures and itsenzymatic activity was characterized using the microsomal fraction ofrecombinant yeast (Sasaki et al., 2008). Sequence homology to SfN8DT-1was the basis for discovery of several other flavonoidprenyltransferases, such as SfiLDT and SfG6DT in S. flavescens (Sasakiet al., 2011) and LaPT1 in Lupinus albus (Shen et al., 2012). Ourprevious work had indicated that resveratrol prenyltransferase(s) inpeanut are membrane-bound proteins that utilize DMAPP from the plastidicterpenoid pathway as the prenyl donor (Yang et al., 2016). These two keyfeatures are also observed in flavonoid-specific prenyltransferasesidentified from other legume species (Sasaki et al., 2008; Akashi etal., 2008; Sasaki et al., 2011; Shen et al., 2012; Li et al., 2014; Chenet al., 2013), suggesting that the sequence of stilbenoidprenyltransferase(s) may share similarity with flavonoidprenyltransferases genes.

To discover and clone peanut prenyltransferase genes, therefore, wefirst built a transcript sequence reference from RNA of our elicitedhairy root culture system, and annotated likely candidate stilbenoidprenyltransferase transcripts by alignment to well-characterizedflavonoid prenyltransferases. An RNA sequencing experiment was designedto capture mRNA temporally associated with stilbene accumulation in thehairy root cultures. We assembled and evaluated a variety of transcriptsequences from RNA-Seq (Mortazavi et al., 2008) reads sets (see Methods)and initially considered any whose translated product aligned tocharacterized flavonoid prenyltransferase sequences listed above. 224transcripts from our set of 2,591,753 transcript assemblies encodedfull-length protein sequences of 101 to 432 amino acid residues thataligned to the set of flavonoid prenyltransferase sequences over alength of at least 100, with >80% sequence identity. As derived from atetraploid, we anticipated that these sequences would representtranscripts of unique enzyme genes, as well as homeologs, alleles andpotential assembly errors. The cultivated peanut genome isallotetraploid thought to have been formed as the result of a singlehybridization between two closely-related diploid species, Arachisduranensis and Arachis ipaensis. Reference sequence assemblies of thelatter diploid genomes were reported recently (Bertioli et al., 2016),and these provided a draft proxy reference we used to evaluate andpotentially reduce our transcriptome to unique genic loci.

Our previous work had implicated prenyltransferase activities ofinterest occurred in the plastid (Yang et al., 2016). We thereforereduced the candidate transcript sequences to ten whose encoded proteinscontain predicted chloroplast targeting peptide sequences according toanalyses with both ChloroP (Emanuelsson et al., 1999) and iPSORT (Bannaiet al., 2002). All ten transcripts showed expression in the 9-hourelicitor-treated hairy root transcriptome, and therefore we used 9-hourelicited hairy roots to clone their cDNAs. PCR using primers designedagainst the assembled transcript sequences resulted in amplification ofthree full-length cDNAs (AhR3′DT-1, AhRPT-10a4 and AhRPT-10d4) encodingstilbenoid prenyltransferase candidates (FIG. 63). We subcloned thesecandidates into yeast expression vector pPICZ, for heterogeneousexpression in Pichia pastoris. The microsomal fractions of the yeastcultures were employed for prenyltransferase assays. However,prenylation activity using resveratrol as substrate was not detected inany of the recombinant yeast cultures assayed (data not shown). Inprevious studies, researchers failed to detect flavonoidprenyltransferase activity when the full-length open reading frame (ORF)of GmG4DT was expressed in yeast, while the truncated form of GmG4DTwithout its N-terminus transit peptide showed geni steinprenyltransferase activity (Yazaki et al., 2009). Likewise, themicrosomal fraction of yeast expressing a truncated form of LaPT1, inwhich the first 44 amino acids were deleted, showed 6-fold higheractivity than that of the full-length protein (Shen et al., 2012).Reasons for these observations may be low tolerance of plant transitpeptides in yeast, correlated with incorrect folding and decreasedstability of the prenyltransferases, resulting in low enzymaticactivity. Rather than removing N-terminal putative transit peptidesequences and reexamining the yeast expression system as in theflavonoid prenyltransferase studies mentioned, we shifted to aheterologous plant expression system. We subcloned the full length cDNAsof the three candidate stilbenoid prenyltransferase into binary vectorsunder the control of CaMV35S-TEV promoter and transiently expressedthese by Agrobacterium-infiltration of Nicotiana benthamiana leaves. Thecrude cell extract of N. benthamiana leaves was incubated with DMAPP andresveratrol to test prenyltransferase activities. One of the cDNAproducts (amplified with primers PT-10-FW-NotI/PT-k-RV-KpnI, FIG. 66)showed resveratrol dimethylallyltransferase activity (FIG. 35). Noactivity was observed in the crude cell extract of N. benthamianacontrol leaves that were infiltrated with Agrobacterium harboring anempty binary vector (FIG. 35). Mass spectrometry analysis of thereaction product (m/z 297 [M+H]⁺) gave a primary fragment with a m/z 241[M+H-56]⁺ in MS² which suggested the presence of a prenyl moiety (FIG.42; FIG. 67). After recovery of the reaction product from a large-scaleenzymatic assay, it was purified by semi-preparative HPLC and itsstructure was further elucidated by NMR analysis.

Because the purified compound was available in extremely low quantities,we continued performing each experiment (¹H-¹³C HMBC & HSQC) over longertime intervals with an increased number of scans. Data obtained showedwell-resolved peaks, forming the basis for our unambiguous assignment ofboth ¹H and ¹³C chemical shifts. In both the ¹H and ¹³C spectra, peaksassigned were in agreement with the original scaffold of resveratrol. Todetermine the position of the prenyl group on the resveratrol scaffold,NMR predict tool was used to generate multiple ¹H and ¹³C spectra forwith various combinations of prenyl positions, with reference to the4-hydroxyphenyl ring (Banfi and Patiny, 2008; Castillo et al., 2011).Two possible prenyl positions appear to be of highest probability andmatched with the experimentally obtained ¹H and ¹³C NMR spectra (FIGS.43A and 43B). We further narrowed down to the final conformation byexamining the ¹H signals at 6.12 ppm, 6.38 ppm, 6.95 ppm, 7.04 ppm, 7.25ppm, 7.49 ppm and were found to be consistent with the resveratrolscaffold. 1H peaks at 3.21 ppm and 5.75 ppm with matched well with theprenyl side chain. (FIG. 43A). The experimental NMR data stronglysupport the predicted location of the prenyl at the ortho position ofthe 4-hydroxyphenyl ring. The conformation of resveratrol is furthercorroborated by the 2D NMR data which show ¹³C peaks at 104.5 ppm, 122.8ppm and 129.0 ppm (FIG. 43B). From the ¹H-¹³C HSQC and ¹H-¹³C HMBCspectrum, the ¹H signal at 7.25 ppm, 6.95 ppm and 6.38 ppm correlatewith protons at the ortho position on 1,3-benzenediol, ethenyl protonand ortho-positioned proton on 4-hydroxyphenyl ring, respectively. Theircorresponding ¹³C signals were at 127.0, 124.2 ppm, and 125.3 ppm,respectively (FIGS. 43C and 43D). Analysis of the NMR data suggest thatthe prenyl moiety is attached to the C-3′ position of the resveratrolbackbone to produce 3-methyl-2-butenyl-3′-resveratrol. Thus, the enzymethat catalyzed this reaction was named as AhR3′DT-1 (A. hypogaearesveratrol-3′-dimethylallyltransferase).

Interestingly, this prenylated resveratrol was not the arachidin-2 wehad expected, given our previous work identifying prenylated products inthe reaction using microsomal fraction of elicited peanut hairy root(Yang et al., 2016). Moreover, the 3-methyl-2-butenyl-3′-resveratrolreaction product described here was not observed in the elicited hairyroot culture of peanut. Our results indicate then that there are otherprenyltransferase(s) responsible for prenylating resveratrol toarachidin-2 in peanut. To further search for additional resveratrolprenyltransferase(s) from the peanut hairy root transcriptome, otherprimer pairs were designed based on the alignment of the putativeprenyltransferase transcripts (FIG. 66). Using this approach, additionalPCR amplicons were amplified from the cDNA of 9-hour-elicited peanuthairy roots and five were subsequently subcloned into binary vectors andtransiently expressed in N. benthamiana leaves for prenyltransferaseactivity assays using DMAPP and resveratrol as substrates. Fouradditional cDNAs were identified as resveratrol prenyltransferase genes.One cDNA clone (amplified with primers PT-9-FW-NotI/PT-b-RV-KpnI, FIG.66) showed a clear dimethylallyltransferase activity for resveratrol andprenylated it at the C-4 position to form arachidin-2 (FIG. 35). Thisproduct was confirmed by comparison of its retention time, UV lightspectrum, mass spectra, and fragmentation patterns obtained by tandemmass spectrometry (MS² and MS³) with arachidin-2 purified from peanuthairy root culture (FIG. 44). Hence, this enzyme was designated asAhR4DT-1 (A. hypogaea resveratrol-4-dimethylallyltransferase). The otherthree cDNA clones (two amplified with primers PT-4-FW-NotI/PT-e-RV-KpnIand one amplified with primers PT-5-FW-NotI/PT-m-RV-KpnI, FIG. 66)exhibited the same catalytic activities as AhR3′DT-1, convertingresveratrol into 3-methyl-2-butenyl-3′-resveratrol using DMAPP as prenyldonor. Hence, we named these AhR3′DT-2, AhR3′DT-3 and AhR3′DT-4. Amongthese four isoenzymes, AhR3′DT-1 exhibited the highest activity.AhR3′DT-2 and AhR3′DT-3 exhibited activity levels that were 18% and 17%of AhR3′DT-1 respectively, while AhR3′DT-4 reached only 5% that ofAhR3′DT-1 (FIG. 35).

Genomic and Phylogenetic Relationships of the Prenyltransferases

We report here five active resveratrol prenyltransferases identifiedfrom biochemical analyses of eight peanut transcripts. Transcriptsassembled from RNA-Seq reads and cloned as cDNAs each independentlyevidence that these encode polypeptides of 389-414 amino acids (FIG.36). All possess nine transmembrane α-helices as predicted by TMHMM 2.0(Krogh et al., 2001) (FIGS. 4 to 7), as well as two aspartate-richmotifs, NQXXDXXXD in loop 2 and KD(I/L)XDX(E/D)GD in loop 6, that arealso conserved in flavonoid prenyltransferases (FIG. 36).

The gene structure of peanut prenyltransferases was estimated byaligning the transcripts to available Arachis diploid progenitor genomesequence references (Bertioli et al., 2016). Four loci in each becameapparent as candidate origins. Three of these were on pseudochromosome 8in each genome, contained within a span containing notable gaps anddescribed by Bertioli et al. (2016) as effected by a genomic reductionduring polyploidization. Only AhR3′DT-4 aligned completely topseudochromosome 1 (FIG. 63). Eliminating poor alignments in eitherprogenitor, we estimated two loci in A. duranensis and two in A.ipaensis could explain the origin of the set of prenyltransferasescharacterized in this study (FIG. 63). Transcript alignments showed thatAhR3′DT-2 and -3 differ by a deletion in AhR3′DT-2 that encodes 16 aminoacid residues in AhR3′DT-3 (FIG. 36). Although the genomic referencecontained gaps in this region that prevented gene structure validation,we expect that these two transcripts may be expressed from the same genein A. hypogaea as alternatively spliced forms (FIG. 63).

Among the eight peanut transcripts tested in N. benthamiana transientassays, three cDNAs failed to show resveratrol prenyltransferaseactivity. One appears to be alternative splice form or allele ofAhR4DT-1, and two of AhR3′DT-1. Observed variation among these mayprovide preliminary insights into structural requirements for the activeforms characterized here. Interestingly, each of the three inactivecDNAs vary in length and sequence at the C-terminal end (45 and 46). Anine amino acid residue deletion at the C-terminus of the inactiveAhR4DT-1 transcript, AhRPT-9i2 likely reduces its encoded proteinstructure to eight transmembrane spans (FIG. 45), suggesting that theintegrity of the nine transmembrane domains in AhR4DT-1 are essentialfor its activity. Two inactive transcripts of AhR3′DT-1 encode aC-terminal extension that does not appear to have transmembraneproperties (FIG. 46). Each furthermore harbors several coding SNPs andan eight amino acid residue deletion (Δ41-48) that disrupts a regionconserved in both active AhR4DT-1 and AhR3′DT-1 (FIG. 36).Interestingly, although not fully deleted in AhR3′DT-2, -3 and -4, thissequence is highly variable in these three less active forms, suggestingthis uncharacterized region may play a role in influencing theprenyltransferase active site.

Phylogenetic analysis of these characterized prenyltransferase enzymestogether showed that the peanut stilbenoid prenyltransferases form theirown monophyletic group (FIG. 37). This clade is notably distinct fromthe flavonoid prenyltransferases, as well as from homogentisate (HG)prenyltransferases involved in ubiquinone and shikonin biosynthesis, andp-hydroxybenzoate (PHB) prenyltransferases involved in the tocotrienol,tocopherol and plastoquinone biosynthetic pathways (FIG. 37).

Biochemical Characterization of AhR4DT-1 and AhR3′DT-1

AhR3′DT-1, the enzyme with the highest activity among its group, alongwith AhR4DT-1 were selected for further biochemical characterization.Similar to the previous resveratrol prenyltransferase activitycharacterized from peanut hairy roots (Yang et al., 2016), the specificactivity in the microsomal fraction of N. benthamiana leaves expressingAhR4DT-1 or AhR3′DT-1 were 8.9-fold and 13.9-fold higher than that inthe crude cell-free extracts, respectively (FIG. 64). Therefore, we usedthe microsomal fraction enriched with AhR4DT-1 or AhR3′DT-1 forsubsequent enzymatic assays. Reactions were incubated with resveratrol,DMAPP and Mg²⁺ as cofactor. Although the optimum activities of AhR4DT-1and AhR3′DT-1 were observed at 37° C. and 30° C., respectively, in themicrosomal fraction of N. benthamina, all further prenylation assayswere performed at 28° C. which corresponds to the culture temperature ofpeanut hairy roots in order to observe the actual behavior of AhR4DT-1and AhR3′DT-1 during the elicitor-induced production of prenylatedstilbenoids (FIG. 49). The accumulation of the prenylated productproduced by AhR4DT-1 or AhR3′DT-1 showed a linear relationship with theamount of the microsomal fraction (25˜75 μg) and with incubation time(30˜120 min) (FIG. 50).

The effects of pH on AhR4DT-1 and AhR3′DT-1 activities were investigatedusing three buffers which spanned a pH range from 7.0 to 10.7. Theoptimum pH of AhR4DT-1 activity was 9.4 in Glycine-NaOH buffer (relativeactivity of each prenyltransferase, 100%), 9.0 in Tris-HCl buffer(99.8%) and 9.7 (92.1%) in NaHCO₃—Na₂CO₃ buffer (FIG. 51A). AhR3′DT-1exhibited optimal activities at pH 9.0 in both Tris-HCl (relativeactivity, 100%) and Glycine-NaOH (92.8%) buffers, but at pH 9.7 inNaHCO₃—Na₂CO₃ buffer (50.6%) (FIG. 51B). In summary, for these twoprenyltransferases, the optimum pH was approximately 9.0, which isconsistent with the basic pH of the stroma (Hauser et al., 1995).Therefore 100 mM of Tris-HCl (pH 9.0) was used as a standard reactionbuffer in subsequent prenyltransferase reactions.

A variety of divalent cations other than Mg²⁺ were tested to determinetheir effects on AhR4DT-1 and AhR3′DT-1 activities. Mg²⁺ was the mosteffective (100%) for AhR4DT-1, followed by Mn²⁺ (83.9%) and Fe²⁺ (6.4%)(FIG. 52A). Interestingly, in AhR3′DT-1 reactions, Mn²⁺ (210.3%)provided 2.1-fold higher efficiency than Mg²⁺ (100%). Mg²⁺ forms abidentate complex with DMAPP which gets stabilized for an efficienttransferase reaction. A possible reason why Mn²⁺ exhibited a higherefficiency could be due to the larger ionic radius facilitating astronger interaction with the pyrophosphate and the neighboring residuesin the DMAPP binding pocket. Fe²⁺ (34.8%) also appeared to contribute toAhR3′DT-1 activity (FIG. 52B). Trace amounts of AhR4DT-1 and AhR3′DT-1activities (<0.5%) were also detected with all other divalent cations(Ca²⁺, Co²⁺, Zn²⁺, Ni²⁺, and Cu²⁺) and in the control group in which nodivalent cation was added, while no activity was detected in the EDTAtreated group (FIG. 52). The trace activities observed are likely due topresence of low levels of Mg²⁺ in the leaf microsomal fractions,released from chlorophyll-containing plastids.

The apparent K_(m) values of AhR4DT-1 for both resveratrol (99.52±15.11μM) and DMAPP (153.7±27.28 μM) were somewhat similar, and comparablewith those of resveratrol prenyltransferase identified from peanut hairyroot (FIG. 65). In contrast, AhR3′DT-1 exhibited a notably lower K_(m)for resveratrol (17.67±1.601 but a much higher K_(m) for DMAPP(691.4±48.46 μM) (FIG. 65; FIG. 53). Piceatannol, a compound detected inpeanut hairy root culture as another putative substrate for peanutprenyltransferase, provided another contrast. The AhR4DT-1 K_(m) forpiceatannol was 311.4±54.83 while that of AhR3′DT-1 was 50.3±5.509 μM153.7±27.28 μM (FIG. 65; FIG. 53). Importantly, AhR4DT-1 and AhR3′DT-1each exhibited a higher V_(max)/K_(m) value for resveratrol thanpiceatannol, suggesting that both of these prenyltransferases preferresveratrol over piceatannol as substrate.

Substrate Specificity of AhR4DT-1 and AhR3′DT-1

In addition to resveratrol, various other stilbenoids (piceatannol,oxyresveratrol, pinosylvin, pterostilbene and piceid) and flavonoids(naringenin, apigenin and genistein) were used as potential substratesto analyze the prenyl acceptor specificity of AhR4DT-1 and AhR3′DT-1.The results (FIG. 38; FIGS. 54 to 58; FIG. 67) showed that AhR4DT-1 canselectively catalyze piceatannol, pinosylvin and oxyresveratrol intoarachidin-5, chiricanine A and prenylated oxyresveratrol (the positionof the prenyl moiety on the prenylated oxyresveratrol remainsundetermined), respectively. In the reactions of AhR3′DT-1, theprenylated products of piceatannol and prenylated oxyresveratrol wereidentified by HPLC-PDA/ESI-MS^(n) analysis (FIGS. 57 to 58; FIG. 67),although the positions of their prenyl moiety have not been confirmeddue to the insufficient amount of these products for further structuralelucidation. Pterostilbene which has two methoxy groups at the C-3 andC-5 positions along with piceid, a resveratrol glucoside with aglycosidic group at C-3 position, were not prenylated by either AhR4DT-1or AhR3′DT-1 (FIG. 38), suggesting that either or both hydroxyl groupson the C-3 and C-5 of stilbene backbone might be crucial for substraterecognition by AhR4DT-1 and AhR3′DT-1. Interestingly, no prenylatedpinosylvin was produced in the AhR3′DT-1 reaction (FIG. 38C), suggestingthat other than the necessary 3- and 5-hydroxyl groups, a hydroxyl groupat C-4′ position might be an additional requirement for AhR3′DT-1activity. Moreover, neither prenylated flavanone, prenylated flavone,nor prenylated isoflavone was detected in either AhR4DT-1 or AhR3′DT-1reactions when flavonoid was used as substrate, indicating that both ofthese peanut prenyltransferases may be stilbenoid-specificprenyltransferases.

To address the prenyl donor specificity of AhR4DT-1 and AhR3′DT-1, inaddition to DMAPP, other prenyl diphosphates, including isopentenylpyrophosphate (IPP), geranyl pyrophosphate (GPP), farnesyl pyrophosphate(FPP) and geranylgeranyl pyrophosphate (GGPP) were examined withresveratrol as a prenyl acceptor. Neither AhR4DT-1 nor AhR3′DT-1 showedany detectable activity when these prenyl diphosphates were used asprenyl donor, suggesting that both of these prenyltransferases hadstrict specificity for DMAPP (FIGS. 4D and 4E).

Subcellular Localization of AhR4DT-1 and AhR3′DT-1

In evaluating primary structures using available software, we foundconflicting predictions for the subcellular localization of theseenzymes. The iPSORT program predicted a chloroplast transit peptide(cTP) in AhR4DT-1 and a mitochondrial targeting peptide (mTP) inAhR3′DT-1. However, ChloroP and TargetP predictions suggested AhR4DT-1contained neither cTP nor mTP, while AhR3′DT-1 contained an N-terminalcTP. To confirm their subcellular localizations experimentally,AhR4DT-1-GFP and AhR3′DT-1-GFP gene fusion constructs driven by theCaMV35S-TEV promoter were expressed transiently in onion epidermal cellsvia particle bombardment. As a positive control for plastidlocalization, we co-expressed a construct created by Nelson et al.(2007) that features the transit peptide (first 79 amino acids) oftobacco RuBisCO small subunit fused to red fluorescent protein(RS-TP-mCherry). The green fluorescence signals of AhR4DT-1-GFP andAhR3′DT-1-GFP appeared in punctate patterns against organelles in theonion epidermal cells (FIG. 39), patterns highly similar to that of thered fluorescence derived from RS-TP-mCherry (FIG. 39). In contrast,control GFP driven by the Ca35S-TEV promoter was localized throughoutthe cytosol and nucleus (FIG. 39). These results strongly suggest thatAhR4DT-1 and AhR3′DT-1 in peanut are localized to plastids, similar toflavonoid prenyltransferases such as SfN8DT-1, GmG4DT and LaPT1characterized in other plant species (Yazaki et al., 2009; Shen et al.,2012; Sasaki et al., 2008).

Expression of AhR4DT-1 and AhR3′DT-1 in Peanut Hairy Roots duringElicitation

Our previous studies have shown that the accumulation of prenylatedstilbenoids in peanut hairy root culture and the prenyltransferaseactivities from crude cell-free extracts of peanut hairy roots wereupregulated by elicitor treatments and increased with incubation time(Yang et al., 2015, 2016). We therefore hypothesized that the mRNA ofenzymes involved in the prenylation of stilbenoids, i.e. AhR4DT-1 andAhR3′DT-1, may accumulate upon elicitation in peanut hairy roots. Totest this hypothesis, transcript levels of AhR4DT-1 and AhR3′DT-1 duringco-treatment of hairy root cultures with MeJA and CD were quantifiedusing real-time PCR. A rapid up-regulation of AhR4DT-1 was observedafter 0.5-hour post-elicitation, and its expression pattern wasconsistent with the prenyltransferase activities assayed in the samesystem (FIGS. 40A and 40B). Despite an apparent delay in accumulation ascompared to AhR4DT-1, levels of AhR3′DT-1 mRNA increased as well, after48-hour of elicitation in these experiments (FIG. 40C). Whereas qPCRdetects a short, sequence-unique portion of the target mRNA, comparativemapping of RNA-Seq reads can provide a more complete picture ofdifferential expression. Assessment of differential expression ofindividual transcripts in this case is however confounded by thecomplexity of this tetraploid transcriptome, the large target enzymefamily, and lack of A. hypogaea genome sequence reference. Employingavailable sequence references of the peanut diploid progenitors (above),we mapped all assembled A. hypogaea transcripts, both to confirmsingularity of genomic loci and to select transcript sequence of eachprenyltransferase that is most sequence-inclusive to use as referencefor quantification (FIG. 65). FIGS. 6D-G represent sample-normalizedcounts of reads that mapped unambiguously to these transcriptreferences. Not surprisingly, mock treatments (assayed at 9 h and 72 h)that mechanically stimulate the hairy root cultures resulted inincreases in all transcripts. AhR4DT-1 mRNA stood out, however, asaccumulating to 2-3× over control levels early in response to additionof MeJA+CD (FIG. 40D). AhR3′DT-1, on the other hand, among treatmentsassayed, reached its highest levels only 72 h post-elicitation (FIG.40E). AhR3′DT-2/3 and -4 transcripts were clearly detectable by RNA-Seqacross the time course, but did not appear to change in response toelicitation (FIG. 40F-G). These results indicate the activation ofAhR4DT-1 and AhR3′DT-1 genes correlates with stress elicitation inpeanut hairy root tissue, and that the accumulation of mRNA encodingthese two enzymes correlates temporally with their prenyltransferaseactivities observed and with their catalyzed product accumulation. Incontrast, mRNA of similar enzymes we show to exhibit activities that arenot relevant, or less relevant, to arachidin-2 and3-methyl-2-butenyl-3′-resveratrol production in peanut are notnoticeably transcriptionally responsive to the elicitation. As productsattributable to AhR4DT-1 and AhR3′DT-1 activities do not accumulate inpeanut hairy root cultures in response to the control treatments usedhere (Yang et al., 2015), protein expression and/or localization arelikely to be controlled by mechanisms beyond the transcript accumulationobserved.

Discussion

Stilbenoid-Specific Prenyltransferases from Peanut

Prenylation of aromatic compounds plays an important role in thediversification of plant secondary metabolites and contributes to theenhancement of the biological activity of these polyphenolic compounds(Yazaki et al., 2009). To date only a few flavonoid prenyltransferasegenes have been identified, including SfN8DT-1, SfiLDT, SfG6DT, SfFPT,GmG4DT, GuA6DT and LaPT1 cloned from legume species (Sasaki et al.,2008, 2011; Chen et al., 2013; Yazaki et al., 2009; Li et al., 2014;Shen et al., 2012), along with MaIDT and CtIDT from non-legume speciesMorus alba and Cudrania tricuspidata, respectively (Wang et al., 2014).In the study reported here, we tested eight potential resveratrolprenyltransferase transcripts and five of them encoded enzymes with twodistinct prenylation activities. The eight transcripts derived from fouror more genes expressed in peanut hairy root cultures. Two of these,AhR4DT-1 and AhR3′DT-1, were characterized as stilbenoid-specificprenyltransferases.

The stilbenoid prenyltransferases described in this study share severalcommon features with flavonoid prenyltransferases. First, each of theseenzymes is a membrane-bound protein containing several putativetransmembrane α-helices. The subcellular localization of the twostilbenoid prenyltransferases described here are primarily orexclusively in the plastid, as are the five flavonoid prenyltransferasespreviously characterized. Secondly, each enzyme contains two conservedaspartate-rich motifs. The observed divalent cation dependency of ourprenylation reactions corroborates the proposed role of this structurein the active site, where the divalent cation and the prenyl diphosphatebind (Huang et al., 2014). Interestingly, with the exception ofAhR3′DT-1 in which Mn²⁺ is most effective, all other flavonoid enzymesand the stilbenoid enzyme AhR4DT-1 show highest activity in the presentof Mg²⁺. Lastly, most flavonoid prenyltransferases and the stilbenoidprenyltransferases identified in this study exhibit strict substratespecificity with respect to their prenyl acceptor and prenyl donor, afeature that contrasts sharply with the catalytically promiscuousaromatic prenyltransferases of fungi and bacteria.

Despite sharing key features with flavonoid prenyltransferases, thestilbenoid prenyltransferases are monophyletic to other plantprenyltransferases accepting aromatic substrates (FIG. 40).

Involvement of AhR4DT-1 and AhR3′DT-1 in the Biosynthesis of PrenylatedStilbenoids in Peanut

AhR4DT-1 specifically transfers a 3,3-dimethylallyl group to the A-ringat the C-4 position of resveratrol, piceatannol and pinosylvin. Thisenzyme exhibits biochemical properties that match well with theprenyltransferase activity identified from elicited peanut hairy roots(Yang et al., 2016), including Km values of resveratrol/DMAPP andidentical preferences for prenyl acceptors and divalent cations. Theconsistency of all biochemical characteristics, along with ourdemonstration of transcript accumulation that is temporally correlatedwith C-4 prenylated stilbenoid (arachidin-1, arachidin-2, arachidin-3and arachidin-5) accumulation, lead us to propose AhR4DT-1 isresponsible for the prenylation activity in the microsomal fraction ofpeanut hairy roots identified in our previous study (Yang et al., 2016).

The second stilbenoid specific prenyltransferase characterized here wasAhR3′DT-1, that recognizes 3,5,4′-trihydroxystilbene and adds a3,3-dimethylallyl group to C-3′ of the B-ring. Notably, none of theprenylation products of resveratrol and piceatannol catalyzed byAhR3′DT-1 were detected in peanut hairy root culture or peanut hairyroot tissue. When compared to AhR4DT-1, AhR3′DT-1 showed a lower Km forresveratrol and piceatannol, indicating a higher affinity for theseprenyl acceptor substrates. In contrast, its affinity for DMAPP was muchlower than that of AhR4DT-1. Furthermore, the V_(max) values ofresveratrol, piceatannol and DMAPP for AhR4DT-1 were 9.9-fold, 6.9-foldand 5.8-fold higher than that of AhR3′DT-1, respectively (FIG. 65),indicating that the catalytic efficiency of AhR4DT-1, especially forutilizing DMAPP, is much higher than that in AhR3′DT-1. This might be areason that no prenylation product of AhR3′DT-1 was found in the peanuthairy root culture, even when the substrate for AhR3′DT-1 was present.

Under the co-treatment of 100 μM MeJA and 9 g/L CD for 72 hours, thepeanut hairy root cultures secrete into the medium large amounts ofresveratrol (44.64±10.26 mg/L), arachidin-1 (77.85±21.47 mg/L) andarachidin-3 (184.65±22.29 mg/L), relatively moderate levels ofarachidin-5 (15.54±5.58 mg/L) and arachidin-2 (25.57±2.98 mg/L), andeven less piceatannol (4.02±0.67 mg/L) (Yang et al., 2015, 2016). Theseobservations suggest that arachidin-1 and arachidin-3 may be endproducts during the tested period of elicitation. Differing fromarachidin-5, arachidin-2, and most other prenylated flavonoids whichharbor a 3,3-dimethylallyl moiety, arachidin-1 and arachidin-3 have aunique 3-methyl-but-1-enyl moiety (FIG. 41). Until now, the biosynthesispathway(s) of arachidin-1 and arachdin-3 have not been fully elucidated,however several biosynthetic routes leading to their production could beproposed when considering results from our previous and current studies.

In our previous study, it was demonstrated that exogenous resveratrolcould be oxidized to piceatannol by an extract from the peanut hairyroot tissue through a very efficient enzymatic reaction (Yang et al.,2016). With the abundance of resveratrol in the culture medium of peanuthairy root, piceatannol generated from the oxidation of resveratrolcould serve as a precursor, alternative to resveratrol, for prenylatedstilbenoids in peanut. It appeared that this compound could be furthermetabolized into other derivatives, resulting in a relative low yield ofpiceatannol in the peanut hairy root culture.

AhR4DT-1 identified here initiates the first step in the biosynthesis ofprenylated stilbenoids in peanut by catalyzing the prenylation ofresveratrol and piceatannol to form arachidin-2 and arachidin-5,respectively. Other than derived that from the prenylation ofpiceatannol, which is limited in the culture medium, it remains possiblethat arachidin-5 is also formed via hydroxylation of arachidin-2 by astilbenoid monooxygenase (P450). Flavonoid 3′-monooxygenases, forexample, are known to catalyze 3′-hydroxylation of the flavonoidbackbone (Tanaka and Brugliera, 2013). Similarly, arachidin-3 might behydroxylated by a monooxygenase to produce arachidin-1 (FIG. 41).Further enzyme discovery and testing are needed to explore thesepossibilities.

In the reactions with peanut hairy root microsomes, arachidin-2 andarachidin-5 prenylated from resveratrol and piceatannol, respectively,could be further converted into derivatives of arachidin-2 derivativeand arachidin-5 that were detected in the medium upon the elicitortreatment (Yang et al., 2016). Nonetheless, these derivatives were notdetected in the AhR4DT-1 reactions (FIG. 41), indicating other enzyme(s)that modify arachidin-2 and arachidin-5 are present downstream ofAhR4DT-1.

In one proposed pathway for arachidin-1 and arachidin-3, arachidin-2 andarachidin-5 might be directly converted to arachidin-3 and arachidin-1by an isomerase which could shift the olefinic bond position on theirprenylated moieties (FIG. 41). Alternatively, arachidin-2 or arachidin-5might be converted into an intermediate product which is furthermodified into arachidin-3 or arachidin-1 through multiple enzymaticsteps (FIG. 41). It is still unclear whether arachidin-2 derivative andarachidin-5 derivative found in the peanut hairy root culture were oneof these intermediates involved in the biosynthesis of arachidin-3 andarachidin-1, which are the predominant compounds in the culture (FIG.41). Alternatively, it may still be a slight possibility thatarachidin-3 and arachidin-1 were directly synthesized from resveratroland piceatannol catalyzed by AhR4DT-1 or another specificprenyltransferase utilizing 3-methyl-but-1-enyl pyrophosphate as prenyldonor. Although, as far as we know, this kind pyrophosphate has neverbeen described in plants.

To date, over 45 prenylated stilbenoids and derivatives, includingmonomers and dimers, have been identified in peanut. Many of thesechemical structures have been confirmed by NMR (FIG. 41) or predicted bymass spectrometry. Interestingly, all these stilbenoids can be dividedinto two groups, one showing a prenyl unit or a derivative at the C-4and a second group showing a prenyl unit or derivative at the C-3′position (FIG. 41). These observations strongly suggest that theprenyltransferases encoded by AhR4DT-1 and AhR3′DT-1 act in the firstcommitted steps that channel the diversification of non-prenylatedstilbenoids into prenylated stilbenoids. As the firststilbenoid-specific prenylated transferases identified in a plant, thesefindings advance our understanding of this specialized gene family andthe biosynthesis of important bioactive compounds in plant stressresponses.

Methods

Plant Materials and Chemical Reagents

Hairy root of peanut cv. Hull line 3 was was previously established bytransforming peanut cotyledonary leaves with Agrobacterium rhizogenesstrain ATCC 15834 and maintained in modified Murashige and Skoog (MSV)medium under continuous darkness at 28° C. as described before (Condoriet al., 2010). The procedure of elicitation for stilbenoids productionin peanut hairy root culture was performed according to Yang et al.,(2015).

Authentic standards of resveratrol and piceatannol were obtained fromBiophysica and Axxoram, respectively. Arachidin-1, arachidin-2,arachidin-3 and arachidin-5 standards were purified from elicited peanuthairy root cultures as described previously (Yang et al., 2016).Pinosylvin, oxyresveratrol, pterostilbene, naringenin, apigenin,genistein, and IPP, GPP, FPP and GGPP were purchased from Sigma-Aldrich.DMAPP used in this study was obtained from Isoprenoids.

RNA Preparation

A co-treatment time-course of 100 μM methyl jasmonate (MeJA; sigma) and9 g/L (6.87 mM) methyl-β-cyclodextrin (CD; Cavasal W7M) was applied tonine-day-old peanut hairy roots to induce the expression of genesinvolved in the biosynthesis of stilbenoids. Total RNA was extractedfrom elicited root tissue at 0.5, 3, 9, 18, 24 and 72 hours using TRIzolreagent (Life Technologies), according to the manufacturer'sinstructions. For controls, RNA was likewise extracted from culturesprior to treatment (t=0) and from non-elicited cultures, collected 9 and72 hours after mock treatment, being refreshed with fresh MSV medium.

Transcript Sequencing and Assembly

Strand-aware, polyA-enriched RNA libraries were prepared using IlluminaTruSeq Stranded mRNA Sample Preparation reagents with sequence-indexedadaptors, with inputs of 4 micrograms total RNA per sample. Averageinsert size of indexed libraries was 300 bp according to Bioanalyzer(Agilent) evaluation. Libraries were sent to the Roy J. CarverBiotechnology Center/W. M. Keck Center at the University of Illinois atUrbana-Champaign, where they were quantified by qPCR and pooled togetherfor sequencing of 2×101 paired cycles on an Illumina HiSeq2500 usingTruSeq SBS sequencing kits version 3. Number of read pairs ranged from34.8 to 58.9 M per sample. Data were processed and demultiplexed usingCasava 1.8.2 (Illumina).

Reads were trimmed using Trimmomatic v 0.32 (Bolger et al., 2014) withheadcrop 12, sliding window 5, minimum quality 25. Parallel assembliesof each sample-specific reads set as well as a combination of all readswere generated using Trinity v 2013-11-10 (Haas et al., 2013),TransABySS 1.5.5 (Robertson et al., 2010), Velvet-Oases 1.2.10 (Schulzet al., 2012) and SOAPtrans 1.03 (Li et al., 2009), yielding a total of2.6 M putative complete and partial transcript sequences. Codingsequences (CDS) were predicted and translated using CD-Hit (Li andGodzik, 2006) through scripts of the Evigenes pipeline (Don Gilbert,Indiana University) to build the transcriptome BlastP and BLASTNdatabases.

Using GMAP 2016-04-04 (Wu and Watanabe, 2005), transcript assemblieswere aligned independently to the Arachis duranensis and Arachisipaensis genomes available in PeanutBase (www.peanutbase.org andBertioli et al., 2016), which confirmed clustering of highly similarforms and allowed us to approximate a reduced A. hypogaea sequencereference against which we could quantify reads coverage attributable tothe enzyme transcripts under study. Reads mapped to the resulting fourgenomic loci were then isolated using Samtools 1.3.1 (Li et al., 2009b)and re-mapped to A. hypogaea transcript references determined here.Mapping of RNA-Seq reads was performed using Tophat2, v 2.0.7 (Kim etal., 2013) using the genome guided option. Uniquely mapped read countsfrom each sample were assessed using HTSeq v 0.6.1p1 (Anders et al.,2015).

Cloning of AhR4DT-1 and AhR3′DT-1 cDNA

The amino acid sequences of flavonoid prenyltransferases SfN8DT-1(accession number: BAG12671.1), SfG6DT (BAK52291.1), SfFPT (AHA36633.1),GmG4DT (BAH22520.1), GuA6DT (AIT11912.1) and LaPT1 (AER35706.1) wereused as input to run BlastP (Altschul et al., 1990) against ourtranslated peanut hairy root transcriptome sequence database.Predictions of chloroplast transit peptides (cTP) were made using bothChloroP (http://www.cbs.dtu.dk/services/ChloroP/) and iPSORT(http://ipsort.hgc.jp/). To clone the full-length cDNA of thesecandidates, RNA of 9-hour-elicited peanut hairy roots was prepared byTRIzol reagent and the cDNA was synthesized using iScript Select cDNASynthesis kit (Bio-Rad) using oligo (dT) primer. One N-terminal primerand three C-terminal primers were synthesized with flanking NotI andKpnI restriction sites, respectively (FIG. 66) and PCR using theseprimers was performed with ExTaq DNA Polymerase (Takara) following theprogram below: initial denaturation (3 min, 94° C.); 30 cycles (30s, 94°C.; 30s, 52° C.; 1 min 30s, 72° C.); and a final extension step (10 min,72° C.). Three amplicons (including AhR3′DT-1) were subcloned into apGEM-T vector (Promega) and multiple clones from each amplicon weresequenced at University of Chicago Comprehensive Cancer Center (UCCCC).In the second screening, according to all the 108 candidate sequences,four N-terminal primers and five C-terminal primers with NotI/KpnIflanking restriction sites (FIG. 66) were designed for prenyltransferasecloning and another four amplicons (including AhR4DT-1) obtained fromthe same cDNA template were cloned into pGEM-T vector for sequencingvalidation.

Phylogenetic Analysis

Protein sequences were aligned using MUSCLE (Edgar 2004), and aneighbor-joining phylogenetic tree computed with PhyML (Guindon andGascuel 2003) using the Dayhoff substitution model and 100 bootstrappedreplicates.

Construction of Binary Vectors

The cloning strategy of binary vectors is showed in FIG. 59. In detail,the sequence of the double enhanced cauliflower mosaic virus 35Spromoter (CaMV35S) fused to the translational enhancer from tobacco etchvirus (TEV) was amplified from plasmid pR8-2 (constructed byMedina-Bolivar and Cramer, 2004) and subcloned into pGEM-T vector usingCa35S-FW-SalI-1/TEV-RW-NotI primers (FIG. 66) with SalI/NotI flankingrestriction sites. After validation of the sequences, thepGEM-CaMV35S-TEV and pGEM-T vectors containing putativeprenyltransferase gene (PT) were digested with SalI/NotI and NotI/KpnI,respectively. A high copy number vector, pBC KS(−) digested withKpnI/SalI was used as a transition vector. Two fragments of full-lengthcDNA and CaMV35S-TEV promoter were ligated into the transition vector ina 16° C. overnight reaction with T4 ligase (NEB). Then the fragment ofCaMV35S-TEV-PT from the transition vector digested by KpnI/SalI wassubcloned into a binary vector, pBIB-Kan which was created by Becker(1990). Eventually, the constructed binary vector with the putativeprenyltransferase gene under the control of CaMV35S-TEV chimericpromoter and three prime untranslated region (3′-UTR) of nopalinesynthase from the original pBIB-Kan vector was transformed into A.tumefaciens LBA4404 for stilbenoid prenylation activity screening.

Screening for Stilbenoid Prenylation Activity

The engineered A. tumefaciens was grown in 5 mL of YEP medium,containing 50 mg/L of kanamycin (Sigma-Aldrich) and 30 mg/L ofstreptomycin (Sigma-Aldrich) for antibiotic selection, at 28° C. on anorbital shaker at 200 rpm. After cultivation for 2 days, 5 mL ofbacterial suspension was inoculated into 50 mL of fresh YEP mediumcontaining the antibiotics and allowed to grow for one additional dayunder the same conditions. Bacteria were pelleted by centrifugation,resuspended in 500 mL of induction medium containing 10 mM MgCl₂ with 1mM acetosyringone and incubated for 4 h at 28° C. under 200 rpm orbitalshaking until their OD₆₀₀ reached to a range from 0.5 to 0.6. Before theinfiltration, 0.005% of Tween-20, 0.005% of Triton 100 and 0.005% ofSilwet L-77 were added to bacterial cultures to enhance the efficiencyof transformation in N. benthamiana leaves. Agrobacterium-mediatedvacuum infiltration was performed on 4-week-old N. benthamiana followingthe methodology described previously (Medrano et al., 2009).

After 48 hours of post-infiltration, the “middle tier” of N. benthamianaleaves were harvested for prenylation activity screening. Five grams ofleaf tissue (fresh weight) were grounded and homogenized using a mortarwith pestle in 10 mL of extraction buffer containing 100 mM Tris-HCl (pH7.6) and 10 mM dithiothreitol (DTT). After removing the cell debris bycentrifugation at 12,000×g for 20 min at 4° C., the crude cell-freeextract was obtained by passing the 12,000×g supernatant through a PD-10desalting column (GE Healthcare) equilibrated with 100 mM Tris-HCl (pH9.0) containing 10 mM DTT. The total protein concentration wasdetermined by coomassie protein assay (Thermo Scientific) using bovineserum albumin as standard.

The prenylation reactions contained resveratrol (100 DMAPP (300 μM),MgCl₂ (10 mM) and DTT (5 mM) in a Tris-HCl buffer (100 mM, pH 9.0).After incubation (28° C., 40 min) with the crude cell-free extract of N.benthamiana leaves (5 mg of total protein) in a total volume of 1 mL,the enzyme reaction was terminated by addition of HCl (6 M, 20 μL) andthen the reaction mixture was extracted with ethyl acetate (1 mL). Theethyl acetate extract was dried under nitrogen gas and dissolved in 300μL of methanol. The reaction product was identified and quantified usingHPLC/ESI-MS^(n) analysis as previously described (Yang et al., 2016).The reaction of crude cell-free extract of N. benthamiana leavesinfiltrated with A. tumefaciens harboring the empty pBIB-Kan vector wasused as control.

Enzymatic Characterization of AhR4DT-1 and AhR3′DT-1

In preliminary experiments focused to study the effect ofpost-infiltration period on the prenylation activity in N. benthamiana,leaf tissues of plants which transiently expressed AhR4D-1 wereharvested 24, 48, 72 and 96 hours post-infiltration. Among thesereactions, the AhR4D-1 activity in the crude cell-free extract of N.benthamiana leaves increased with the post-infiltration period from 24to 72 hours, while the activity in the leaves harvested from 96 hourspost-infiltration was similar to 72 hours (data not shown). In addition,since both AhR4DT-1 and AhR3′DT-1 were predicted as membrane boundproteins by TMHMM 2.0, the microsomal fraction of N. benthamiana leavesat 72 hours post-infiltration was used to study the biochemicalproperties of AhR4DT-1 or AhR3′DT-1. Ten grams of fresh leaves werehomogenized using a mortar with pestle in a 20 mL of extraction buffer(100 mM Tris-HCL pH 7.6 containing 10 mM DTT). The homogenate wascentrifuged at 12,000×g for 20 min at 4° C., and about 13.2 mL of thesupernatant was centrifuged at 156,000×g for 45 min at 4° C. to pelletthe microsomal fraction, while the 156,000×g supernatant was prepared byusing a PD-10 desalting column (GE Healthcare) equilibrated withTris-HCl buffer (100 mM, pH 9.0) containing DTT (10 mM). The microsomalfraction was washed twice with Tris-HCl buffer (100 mM, pH 9.0)containing DTT (10 mM) and resuspended in 1 mL of the same buffer.

The basic reaction and measurement for AhR4DT-1 and AhR3′DT-1 activitywere the same as that for prenylation activity screening with exceptionof using 30 μg of microsomal fraction of N. benthamiana leaves as enzymeinstead of crude cell-free extract of N. benthamiana leaves in a 500 μLof reaction. To investigate the optimal pH, the enzymatic reactions wereperformed in Tris-HCl buffer (100 mM, pH 7.0 to 9.0), glycine-NaOHbuffer (100 mM pH 8.6 to 10.6) and NaHCO₃—Na₂CO₃ buffer (100 mM, pH 9.2to 10.7). The optimal reaction temperatures for AhR4DT-1 and AhR3′DT-1were tested at 20, 25, 28, 30, 37, 40, and 50° C. in Tris-HCl buffer(100 mM, pH 9.0). For the divalent cation dependency study, 10 mM MnCl₂,FeCl₂, CaCl₂, CoCl₂, ZnCl₂, NiCl₂, or CuCl₂ was added to the reactionmixture instead of MgCl₂, and the enzyme activity was compared with thereaction containing MgCl₂. The reactions without divalent cation and 10mM EDTA instead of MgCl₂ were used as controls.

For the kinetic study, varying concentrations (10, 20, 40, 80, 160, 320,and 640 μM) of resveratrol or piceatannol with a fixed concentration ofDMAPP (640 μM) and varying concentrations (10, 20, 40, 80, 160, 320, and640 μM) of DMAPP with a fixed concentration of resveratrol (640 μM) wereincubated with 30 μg of microsomal fractions of N. benthamiana leavesexpressing AhR4DT-1 or AhR3′DT-1 to calculate the apparent K_(m) andV_(max) values by nonlinear regression analysis of the Michaelis-Mentenequation using GraphPad Prism 6 software. The prenyl acceptorspecificity of AhR4DT-1 and AhR3′DT-1 were tested using 100 μM of eachstilbenoid (resveratrol, piceatannol, oxyresveratrol, pinosylvin,pterostilbene and piceid), flavanone (naringenin), flavone (apigenin),and isoflavone (genistein) with 300 μM DMAPP as a prenyl donor, whilethe prenyl donor specificities of these two enzymes were tested using300 μM prenyl diphosphates (DMAPP, IPP, GPP, FPP, GGPP) with 100 μMresveratrol as a prenyl acceptor. All these reactions were performed ina total volume of 500 μL with 100 mM Tris-HCl buffer (pH 9.0) at 28° C.for 40 min.

NMR Spectra

All NMR measurements were performed on a Bruker Avance 700 MHz andspectrometers at 298 K. The ¹H-¹³C HMBC and ¹H-¹³C HSQC spectra werecollected in d₆-acetone. The concentration of the sample was ˜1 mM. For¹H NMR analysis, 16 transients were acquired with a 1 second relaxationdelay using 32 K data points. The 90° pulse was 9.7 μs with a spectralwidth of 16 ppm. 1D ¹³C NMR spectra were obtained with a spectral widthof 30 ppm collected with 64 K data points. Two-dimensional spectra wereacquired with 2048 data points for t2 and 256 for t1 increments. All NMRdata were analyzed using Topspin v 2.0 and SPARKY v 3.0 software. Peakswere integrated and overlaid with the simulated spectra for differentversions of the prenyl chain attached on the resveratrol compound

Construction of GFP Fusion Proteins

The nucleotide sequence of modified green fluorescence protein (mGFP5)was amplified from pR8-2 (Medina-Bolivar and Cramer, 2004) using primersmgfp5-FW-BamHI/mgfp5-RW-KpnI (FIG. 66) and cloned into pGEM-T vector togive pGEM-mGFP5-1. PT-9b13-RV-BamHI or PT-10k1-RV-BamHI reverse primerwith Ca35S-FW-SalI-2 forward primer were used to amplify the full-lengthof AhR4DT-1 or AhR3′DT-1 with CaMV35S-TEV promoter region frompBC-CaMV35S-TEV-9b13 and pBC-CaMV35S-TEV-10k1 vector, which were createdduring the construction of the binary vector (FIG. 60; FIG. 68). The PCRproducts were then cloned into pGEM-T vector for sequencing validation.After SalI/BamH1 digestion, CaMV35S-TEV-AhR4DT-1 andCaMV35S-TEV-AhR3′DT-1 fragments were isolated and inserted intopGEM-mGFP5-1 to yield pGEM-CaMV35S-TEV-AhR4DT-1-GFP andpGEM-CaMV35S-TEV-AhR3′DT-1-GFP, respectively. Lastly, the fragments ofCaMV35S-TEV-AhR4DT-1-GFP and CaMV35S-TEV-AhR3′DT-1-GFP were excised withSalI/KpnI and ligated into binary vector pBIB-Kan to yieldpBIBKan-AhR4DT-1-GFP and pBMKan-AhR3′DT-1-GFP, respectively (FIG. 61).For the GFP control construct, mGFP5 gene was amplified from pR8-2 usingprimers mgfp5-FW-NotI/mgfp5-RW-KpnI and cloned into pGEM-T vector togive pGEM-mGFP5-2. Two fragments CaMV35S-TEV digested frompGEM-CaMV35S-TEV by SalI/NotI and mGFP5 digested from pGEM-mGFP5-2 byNotI/KpnI were inserted into pBC KS(−) vector to formpBC-CaMV35S-TEV-GFP. The fragment of CaMV35S-TEV-GFP was eventuallysubcloned into binary vector pBIB-Kan to form pBIB-Kan-GFP (FIG. 61).

Particle Bombardment and Microscopy

To investigate the subcellular localization of AhR4DT-1 and AhR3′DT-1,pBM-Kan-AhR4DT-1-GFP, pBM-Kan-AhR3′DT-1-GFP and pBM-Kan-GFP wereco-bombarded with binary vector pt-rk (ABRC stock numbers: CD3-999,Nelson et al., 2007) containing a plastid marker fused with redfluorescent protein into the onion epidermal peel cells by PDS-1000/He™systems (Bio-Rad) following the manufacturer's recommendations. Inbrief, 5 μg of target plasmid and 5 μg of pt-rk plasmid were togethercoated on 50 μL of 60 mg/mL tungsten particles (M17, 1 μm; Bio-Rad) inthe presence of 1 M CaCl₂ and 15 mM spermidine. After several ethanolwashes, plasmid-coated particles were dried on plastic discs andaccelerated with a helium burst at 1100 psi in a bombardment chamber.Bombarded onion epidermal peels were kept on plates containing MS mediumfor 60 hours in the dark. The localization of the expressed proteins inthe transformed cell was visualized with a Nikon Eclipse E800 microscopewith a 20×/0.5W Fluor water immersion objective. Confocal fluorescenceimages were obtained by using Nikon digital eclipse C1 microscope systemwith 488 nm laser illumination and 525/50 nm filter for GFP fluorescenceand 543 nm laser with 595/50 nm filter for RFP fluorescence.

Quantitative Real-Time PCR of AhR4DT-1 and AhR3′DT-1

Total RNA was isolated from 100 μM MeJA and 9 g/L CD co-treated peanuthairy roots at 0.5, 3, 9, 18, 24 and 72 hours using TRIzol reagent, andcDNA was synthesized using iScript™ Select cDNASynthesis Kit (Bio-Rad)with oligo (dt) primers following the manufacturer's instructions.Primers for AhR4DT-1 and AhR3′DT-1 were designed using Allele ID(PREMIER Biosoft). Two reference genes, ACTT (encoding actin 7) and EFα1(encoding elongation factor α1) were selected previously (Condon et al.,2011) and used to normalize the expression of AhR4DT-1 and AhR3′DT-1 inpeanut hairy roots. Efficiencies of primers for these targets are shownin FIG. 62. Quantitative real-time PCR (qPCR) reactions were carried outusing iQ SYBR Green Supermix (Bio-Rad) as previously described (Yang etal., 2015) and the expression of AhR4DT-1 and AhR3′DT-1 were analyzed byqbase+ (Biogazelle).

Accession Numbers

The nucleotide sequences of AhR4DT-1, AhR3′DT-1, AhR3′DT-2, AhR3′DT-3,AhR3′DT-4 have been deposited in the GenBank™ database under theaccession numbers KY565244, KY565245, KY565246, KY565247 and KY565248,respectively.

Stable Expression of Peanut Stilbenoid-Specific Prenyltransferase inTobacco Plants and Hairy Roots.

To establish transgenic tobacco (Nicotiana tabacum) plants expressingpeanut stilbenoid prenyltransferase genes, pBIB-Kan binary vectorscontaining either the AhR4DT-1 or AhR3′DT-1 cDNA under the control ofthe constitutive 35S promoter were transformed into Agrobacteriumtumefaciens LBA4404. Leaf blade, petiole and stem explants of N. tabacumwere wounded and inoculated with above engineered A. tumefaciens. After48 hours of inoculation, explants were placed in regeneration medium(modified MS medium containing 1 mg/L BAL and 0.1 mg/L NAA), 600 mg/Lcefotaxime and 200 mg/L kanamycin for selection of transgenic plants.After 2-3 weeks, transgenic callus that developed at the inoculationsite were harvested and transferred to medium containing 600 mg/lcefotaxime and 200 mg/L kanamycin, and maintained at 24° C. for another2 weeks for shoot development. Newly developed shoots were transferredto antibiotic-free medium for roots of whole plants (FIG. 11).

To confirm that the prenyltransferase gene was integrated into the plantgenome, genomic DNA was isolated from transgenic lines using DNeasyPlant Mini Kit (QIAGEN). Primer pairs that targeted the outside ofprenyltransferase gene on pBIB-Kan vectors were used for molecularcharacterization of transgenic lines. The transgenic plants transformedwith empty pBIB-Kan vector showed an amplicon of 523 bp, while theAhR4DT-1 and AhR3′DT-1 transgenic lines gave amplicons of 2673 bp and2619 bp, respectively. The AhR4DT-1 or AhR3′DT-1 activity in thesetransgenic lines was further confirmed via enzymatic assay (FIGS. 71Cand 72C). In summary, five AhR4DT-1-expressing tobacco plants and nineAhR3′DT-1-expressing tobacco plants of N. tabacum were established(FIGS. 71 and 72).

Two hairy root lines, line 1 and line 2, were developed from theAhR3′DT-1-expressing tobacco plant #12 via Agrobacterium rhizogenes15834 infection and the activity of the AhR3′DT-1 was detected from bothof these transgenic hairy root lines (FIG. 73).

These results indicate that the peanut stilbenoid-specific transferases(AhR4DT-1 or AhR3′DT-1) were actively functional in the transgenictobacco plants and hairy roots.

Statistical Analysis

Two-way ANOVA with multiple-comparisons tests was conducted for the datain FIG. 3. Analyses were done with GraphPad Prism 6, version 6.02software.

LITERATURE CITED

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From the foregoing, it will be seen that the present invention is onewell adapted to obtain all the ends and objects herein set forth,together with other advantages which are inherent to the structure.

It will be understood that certain features and subcombinations are ofutility and may be employed without reference to other features andsubcombinations. This is contemplated by and is within the scope of theclaims.

As many possible embodiments may be made of the invention withoutdeparting from the scope thereof, it is to be understood that all matterherein set forth or shown in the accompanying drawings is to beinterpreted as illustrative and not in a limiting sense.

What is claimed is:
 1. A method of producing a prenylated stilbenoid ina transgenic organism, cell or tissue, the method comprising: generatinga transgenic organism, cell, or tissue comprising a transgene encoding apolypeptide comprising an amino acid sequence comprising the sequence asset forth in SEQ ID NO: 2 having stilbenoid prenyltransferase activity,and expressing the polypeptide.
 2. A method of producing a prenylatedstilbenoid in a transgenic organism, cell or tissue, the methodcomprising: generating a transgenic organism, cell, or tissue comprisinga transgene comprising a nucleic acid molecule comprising the sequenceas set forth in SEQ ID NO: 3 that encodes a polypeptide havingstilbenoid prenyltransferase activity, and expressing the nucleic acidmolecule.